Inhibitors of protein arginine deiminases (pads) and methods of preparation and use thereof

ABSTRACT

The invention provides novel inhibitors or inactivators of protein arginine deiminases, pharmaceutical compositions and methods of use thereof. The invention also relates to molecular probes based on such compounds and methods of use thereof.

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/429,099, filed Dec. 2, 2016, the entire content of which isincorporated herein by reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. GM079357awarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to therapeutic compounds, pharmaceuticalcompositions and methods thereof. More particularly, the inventionprovides inhibitors or inactivators of protein arginine deiminases,pharmaceutical compositions and methods of preparation and use thereof.The invention also relates to molecular probes based on such compoundsand methods of preparation and use thereof.

BACKGROUND OF THE INVENTION

A significant portion of the general population is affected by immunesystem disorders, which include abnormally low activity or over activityof the immune system. In cases of autoimmune diseases, the body's overlyactive immune system attacks and damages its own tissues. In cases of anoverly inactive immune system, the body's ability to fight invadersdecreases due to immune deficiency, leaving the patient vulnerable toinfections. Currently, treatment for autoimmune diseases focuses onrelieving symptoms because there is no curative therapy.

Immune system disorders, such as rheumatoid arthritis (RA), multiplesclerosis, lupus, and ulcerative colitis, are placing an increasingburden on society, impairing the health and lives of those affected.Although medications have been developed to treat some of these diseasesand conditions, the available treatments are often limited in terms ofclinical effectiveness and at the same time have undesirable sideeffects.

Protein arginine deiminases (PADs) are a unique family of enzymes thatcatalyzes a form of post-translational modification called argininedeimination or citrullination: the hydrolysis of peptidyl-arginine toform peptidyl-citrulline on histones, fibrinogen, and other biologicallyrelevant proteins. The post-translational modification of histones hassignificant effects on overall chromatin function.

The PAD reaction involves the hydrolysis of the guanidinium group ofarginine to generate citrullinated proteins. This reaction is acalcium-dependent process wherein calcium binding triggers aconformational change that moves a nucleophilic cysteine residue intothe active site, resulting in a >10,000-fold increase in PAD activity.Overexpression and/or increased PAD activity is observed in severaldiseases, including rheumatoid arthritis, Alzheimer's disease, multiplesclerosis, lupus, Parkinson's disease, and cancer. (Liu, et al. 2011PLoS One 6, e21314; Kearney, et al. 2005 Biochemistry 44, 10570-10582.)

There are five PAD isozymes (PADs 1-4 and 6) with unique cellular andtissue distribution patterns, where only PADs 1-4 have been isolated intheir catalytically active form. The PADs are uniquely distributed bothwithin the cell and throughout the body. Specifically, all the PADs canbe found in the cellular cytoplasm while only PADs 2, 3 and 4 areexpressed in the nucleus. (Jones, et al. 2009 Curr Opin Drug DiscovDevel 12, 616-627. Vossenaar, et al. 2003 Bioessays 25, 1106-1118;Stone, et al. 2005 Biochemistry 44, 13744-13752; Fuhrmann, et al. 2015Chem Rev 115, 5413-5461; Nakashima, et al. 2002 J Biol Chem 277,49562-49568; Cherrington, et al. 2010 PLoS One 5, el 1768; Jang, et al.2011 J Neuropathol Exp Neurol 70, 116-124; Li, et al. 2016 PLoS One 11,e0147503.)

There is an urgent and growing need for novel therapeutics and treatmentmethods that provide improved clinical effectiveness with reduced sideeffects, in particular through safe and effective inhibition orinactivation of PADs.

SUMMARY OF THE INVENTION

The invention provides novel inhibitors or inactivators of PADs,pharmaceutical compositions and methods of preparation and use thereof.The compounds and pharmaceutical compositions of the invention may beused to treat immune system disorders and inflammatory diseases andconditions (e.g., rheumatoid arthritis, lupus).

The invention also provides novel molecular probes (e.g., imagingprobes) for PADs based on the inhibitors or inactivators of PADsdisclosed herein in conjugation with detectable labels such asfluorescent dyes, and methods of preparation and use thereof. Themolecular imaging probes of the invention may be used to screen oridentify compounds for PAD inhibition or inactivation.

In one aspect, the invention generally relates to a compound having thestructural formula

wherein

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof.

In another aspect, the invention generally relates to a pharmaceuticalcomposition comprising a compound having the structural formula of (I):

wherein,

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof, effective to treat,prevent, or reduce one or more diseases or disorders, in a mammal,including a human, and a pharmaceutically acceptable excipient, carrier,or diluent.

In yet another aspect, the invention generally relates to a unit dosageform comprising a pharmaceutical composition disclosed herein.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition comprising a compound having the structural formula of (I):

wherein,

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof, and a pharmaceuticallyacceptable excipient, carrier, or diluent, effective to treat, prevent,or reduce one or more diseases or disorders, in a mammal, including ahuman.

In yet another aspect, the invention generally relates to a method forinhibiting or inactivating a protein arginine deiminase, comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising a compound having the structural formula of (I):

wherein,

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c) and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof, and a pharmaceuticallyacceptable excipient, carrier, or diluent, effective to inhibit orinactivate a biological function of a protein arginine deiminase, in amammal, including a human.

In yet another aspect, the invention generally relates to a molecularimaging probe having the structural formula:

A_(F)-L_(F)-W   (II)

wherein

A_(F) is a group comprising an optically detectable moiety;

L_(F) is a linking group; and

W is group comprising a benzimidazole moiety, or a derivative or analogthereof, capable of inhibiting or inactivating a biological function ofa protein arginine deiminase.

In yet another aspect, the invention generally relates to a method foridentifying a protein arginine deiminase inhibitor or inactivator. Themethod includes: performing a competitive assay wherein a test compoundcompetes with a molecular imaging probe disclosed herein to bind to aprotein arginine deiminase; and measuring fluorescence to determine anamount of fluorescent protein arginine deiminase present in the testassay.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) Concentration dependent labeling of recombinant PAD2 withBB—F-Yne (5u). PAD2 was treated with increasing concentrations ofBB—F-Yne (5u) and then “Clicked” with TAMRA-N₃. (B) The limit ofdetection (LOD) of BB—F-Yne (5u) for PAD2. Decreasing concentrations ofPAD2 treated with BB—F-Yne (5u) and “Clicked” with TAMRA-N₃. The LOD wasfound to be 375 fmol. (C) Concentration dependent labeling ofrecombinant PAD2 with BB—Cl-Yne (5v). PAD2 was treated with increasingconcentrations of BB—Cl-Yne (5) and then “Clicked” with TAMRA-N₃. (D)The LOD of BB—Cl-Yne (5v) for PAD2. Decreasing concentrations of PAD2were treated with BB—Cl-Yne (5v) and “Clicked” with TAMRA-N₃.

FIG. 2. In vitro labeling of the four active PAD isozymes with BB—F-Yne(5u). (A) Concentration dependent labeling of recombinant PAD1 withBB—F-Yne (5u). PAD1 was treated with increasing concentrations ofBB—F-Yne (5u) and then “Clicked” with TAMRA-N₃. (B) Concentrationdependent labeling of recombinant PAD2 with BB—F-Yne (5u). PAD2 wastreated with increasing concentrations of BB—F-Yne (5u) and then“Clicked” with TAMRA-N₃. This data can also be seen in FIG. 1 and isrepeated here for clarity. (C) Concentration dependent labeling ofrecombinant PAD3 with BB—F-Yne (5u). PAD3 was treated with increasingconcentrations of BB—F-Yne (5u) and then “Clicked” with TAMRA-N₃. (D)Concentration dependent labeling of recombinant PAD4 with BB—F-Yne (5u).PAD4 was treated with increasing concentrations of BB—F-Yne (5u) andthen “Clicked” with TAMRA-N₃.

FIG. 3. Cellular labeling of PAD2 with BB—F-Yne (5u) in ionophorestimulated HEK293T/PAD2 cells. (A) HEK293T/PAD2 cells were treated withincreasing concentrations of BB—F-Yne (5u) for 1 h. The cells were thenharvested and probe labeled proteins were tagged with TAMRA-N₃ tofacilitate visualization after SDS-PAGE. (B&C) HEK293T/PAD2 cells weretreated with increasing concentrations of BB—F-Yne (5u) for 1 h. Thecells were then harvested and probe labeled proteins were tagged withBiotin-TEV-N₃. Biotin tagged proteins were then isolated on streptavidinagarose and the eluted proteins were probed for PAD2 (panel B) orbiotinylated proteins using streptavidin-HRP (panel C).

FIG. 4. Cellular labeling of PAD2 with BB—Cl-Yne (5v) in ionophorestimulated HEK293T/PAD2 cells. (A) HEK293T/PAD2 cells were treated withincreasing concentrations of BB—Cl-Yne (5) for 1 h. The cells were thenharvested and probe labeled proteins were tagged with TAMIRA-N₃ tofacilitate visualization after SDS-PAGE. (B&C) HEK293T/PAD2 cells weretreated with increasing concentrations of BB—Cl-Yne (5v) for 1 h. Thecells were then harvested and probe labeled proteins were tagged withBiotin-TEV-N₃. Biotin tagged proteins were then isolated on streptavidinagarose and the eluted proteins were probed for PAD2 (panel B) orbiotinylated proteins using streptavidin-HRP (panel C).

FIG. 5. Cellular labeling of PAD4 with BB—F-Yne (5u). (A) HEK293T/PAD4cells were treated with increasing concentrations of BB—F-Yne (5u) for 1h. The cells were then harvested and probe labeled proteins were taggedwith TAMIRA-N₃ to facilitate visualization after SDS-PAGE. (B)HEK293T/PAD4 cells were treated with increasing concentrations ofBB—F-Yne (5u) for 1 h. The cells were then harvested and probe labeledproteins were tagged with Biotin-TEV-N₃. Biotin tagged proteins werethen isolated on streptavidin agarose and the eluted proteins wereprobed for PAD2.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. General principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry”, Thomas Sorrell, University ScienceBooks, Sausalito: 2006.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios arecontemplated by the present invention. Those of ordinary skill in theart will readily appreciate that analogous ratios are contemplated formore complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic methods well known in the art, and subsequent recoveryof the pure enantiomers.

As used herein, “administration” of a disclosed compound encompasses thedelivery to a subject of a compound as described herein, or a prodrug orother pharmaceutically acceptable derivative thereof, using any suitableformulation or route of administration, as discussed herein.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” refer to that amount of a compound or pharmaceuticalcomposition described herein that is sufficient to effect the intendedapplication including, but not limited to, disease treatment, asillustrated below.

In some embodiments, the amount is that effective for stop theprogression or effect reduction of an inflammatory disease or disorder.In some embodiments, the amount is that effective for stop theprogression or effect reduction of an immune system disorders. In someembodiments, the amount is that effective to stop the progression oreffect reduction of an autoimmune disease or disorder. In someembodiments, the amount is that effective for stop the progression oreffect reduction of a cardiovascular disease or disorder. In someembodiments, the amount is that effective for detectable killing orinhibition of the growth or spread of cancer cells; the size or numberof tumors; or other measure of the level, stage, progression or severityof the cancer.

The therapeutically effective amount can vary depending upon theintended application, or the subject and disease condition beingtreated, e.g., the desired biological endpoint, the pharmacokinetics ofthe compound, the disease being treated, the mode of administration, andthe weight and age of the patient, which can readily be determined byone of ordinary skill in the art. The term also applies to a dose thatwill induce a particular response in target cells, e.g., reduction ofcell migration. The specific dose will vary depending on, for example,the particular compounds chosen, the species of subject and theirage/existing health conditions or risk for health conditions, the dosingregimen to be followed, the severity of the disease, whether it isadministered in combination with other agents, timing of administration,the tissue to which it is administered, and the physical delivery systemin which it is carried.

As used herein, the terms “treatment” or “treating” a disease ordisorder refers to a method of reducing, delaying or ameliorating such acondition before or after it has occurred. Treatment may be directed atone or more effects or symptoms of a disease and/or the underlyingpathology. Treatment is aimed to obtain beneficial or desired resultsincluding, but not limited to, therapeutic benefit and/or a prophylacticbenefit. By therapeutic benefit is meant eradication or amelioration ofthe underlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient can still be afflicted with the underlying disorder. Forprophylactic benefit, the pharmaceutical compounds and/or compositionscan be administered to a patient at risk of developing a particulardisease, or to a patient reporting one or more of the physiologicalsymptoms of a disease, even though a diagnosis of this disease may nothave been made. The treatment can be any reduction and can be, but isnot limited to, the complete ablation of the disease or the symptoms ofthe disease. As compared with an equivalent untreated control, suchreduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%,60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

As used herein, the term “therapeutic effect” refers to a therapeuticbenefit and/or a prophylactic benefit as described herein. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

As used herein, a “pharmaceutically acceptable form” of a disclosedcompound includes, but is not limited to, pharmaceutically acceptablesalts, esters, hydrates, solvates, isomers, prodrugs, and isotopicallylabeled derivatives of disclosed compounds. In one embodiment, a“pharmaceutically acceptable form” includes, but is not limited to,pharmaceutically acceptable salts, esters, isomers, prodrugs andisotopically labeled derivatives of disclosed compounds. In someembodiments, a “pharmaceutically acceptable form” includes, but is notlimited to, pharmaceutically acceptable salts, esters, stereoisomers,prodrugs and isotopically labeled derivatives of disclosed compounds.

In certain embodiments, the pharmaceutically acceptable form is apharmaceutically acceptable salt. As used herein, the term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of subjects without undue toxicity, irritation,allergic response and the like, and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are well known inthe art. For example, Berge et al. describes pharmaceutically acceptablesalts in detail in J. Pharmaceutical Sciences (1977) 66:1-19.Pharmaceutically acceptable salts of the compounds provided hereininclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchioric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. In some embodiments, organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, lactic acid, trifluoracetic acid,maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, andthe like.

The salts can be prepared in situ during the isolation and purificationof the disclosed compounds, or separately, such as by reacting the freebase or free acid of a parent compound with a suitable base or acid,respectively. Pharmaceutically acceptable salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN+(C₁₋₄alkyl)⁴ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate and aryl sulfonate. Organic bases fromwhich salts can be derived include, for example, primary, secondary, andtertiary amines, substituted amines, including naturally occurringsubstituted amines, cyclic amines, basic ion exchange resins, and thelike, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. In some embodiments,the pharmaceutically acceptable base addition salt can be chosen fromammonium, potassium, sodium, calcium, and magnesium salts.

In certain embodiments, the pharmaceutically acceptable form is apharmaceutically acceptable ester. As used herein, the term“pharmaceutically acceptable ester” refers to esters that hydrolyze invivo and include those that break down readily in the human body toleave the parent compound or a salt thereof. Such esters can act as aprodrug as defined herein. Pharmaceutically acceptable esters include,but are not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, andcycloalkyl esters of acidic groups, including, but not limited to,carboxylic acids, phosphoric acids, phosphinic acids, sulfinic acids,sulfonic acids and boronic acids. Examples of esters include formates,acetates, propionates, butyrates, acrylates and ethylsuccinates. Theesters can be formed with a hydroxy or carboxylic acid group of theparent compound.

As used herein, the term “pharmaceutically acceptable enol ethers”include, but are not limited to, derivatives of formula —C═C(OR) where Rcan be selected from alkyl, alkenyl, alkynyl, aryl, aralkyl andcycloalkyl. Pharmaceutically acceptable enol esters include, but are notlimited to, derivatives of formula —C═C(OC(O)R) where R can be selectedfrom hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl and cycloalkyl.

In certain embodiments, the pharmaceutically acceptable form is a“solvate” (e.g., a hydrate). As used herein, the term “solvate” refersto compounds that further include a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces. Thesolvate can be of a disclosed compound or a pharmaceutically acceptablesalt thereof. Where the solvent is water, the solvate is a “hydrate”.Pharmaceutically acceptable solvates and hydrates are complexes that,for example, can include 1 to about 100, or 1 to about 10, or 1 to about2, about 3 or about 4, solvent or water molecules. It will be understoodthat the term “compound” as used herein encompasses the compound andsolvates of the compound, as well as mixtures thereof.

In certain embodiments, the pharmaceutically acceptable form is aprodrug. As used herein, the term “prodrug” (or “pro-drug”) refers tocompounds that are transformed in vivo to yield a disclosed compound ora pharmaceutically acceptable form of the compound. A prodrug can beinactive when administered to a subject, but is converted in vivo to anactive compound, for example, by hydrolysis (e.g., hydrolysis in blood).In certain cases, a prodrug has improved physical and/or deliveryproperties over the parent compound. Prodrugs can increase thebioavailability of the compound when administered to a subject (e.g., bypermitting enhanced absorption into the blood following oraladministration) or which enhance delivery to a biological compartment ofinterest (e.g., the brain or lymphatic system) relative to the parentcompound. Exemplary prodrugs include derivatives of a disclosed compoundwith enhanced aqueous solubility or active transport through the gutmembrane, relative to the parent compound.

The prodrug compound often offers advantages of solubility, tissuecompatibility or delayed release in a mammalian organism (see, e.g.,Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam). A discussion of prodrugs is provided in Higuchi, T., et al.,“Prodrugs as Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14,and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche,American Pharmaceutical Association and Pergamon Press, 1987, both ofwhich are incorporated in full by reference herein. Exemplary advantagesof a prodrug can include, but are not limited to, its physicalproperties, such as enhanced water solubility for parenteraladministration at physiological pH compared to the parent compound, orit can enhance absorption from the digestive tract, or it can enhancedrug stability for long-term storage.

As used herein, the term “pharmaceutically acceptable” excipient,carrier, or diluent refers to a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject pharmaceutical agent from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations. Wetting agents, emulsifiers and lubricants,such as sodium lauryl sulfate, magnesium stearate, and polyethyleneoxide-polypropylene oxide copolymer as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

Compounds of the present invention are, subsequent to their preparation,preferably isolated and purified to obtain a composition containing anamount by weight equal to or greater than 95% (“substantially pure”),which is then used or formulated as described herein. In certainembodiments, the compounds of the present invention are more than 99%pure.

Solvates and polymorphs of the compounds of the invention are alsocontemplated herein. Solvates of the compounds of the present inventioninclude, for example, hydrates.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. When a range of values is listed, it isintended to encompass each value and sub-range within the range. Forexample “C₁₋₆ alkyl” is intended to encompass, C₁, C₂, C₃, C₄, C₅, C₆,C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆, C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄,C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

As used herein, the term “alkyl” refers to a straight or branchedhydrocarbon chain radical consisting solely of carbon and hydrogenatoms, containing no unsaturation, having from one to ten carbon atoms(e.g., C₁₋₁₀ alkyl). Whenever it appears herein, a numerical range suchas “1 to 10” refers to each integer in the given range; e.g., “1 to 10carbon atoms” means that the alkyl group can consist of 1 carbon atom, 2carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated. In some embodiments,“alkyl” can be a C₁₋₆ alkyl group. In some embodiments, alkyl groupshave 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms. Representativesaturated straight chain alkyls include, but are not limited to,-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; whilesaturated branched alkyls include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl,3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl,2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,2,3-dimethylbutyl, and the like. The alkyl is attached to the parentmolecule by a single bond. Unless stated otherwise in the specification,an alkyl group is optionally substituted by one or more of substituentswhich independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy,alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino,imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy,haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio,thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl,sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,—Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R_(a),—C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or 2), —P(═O)(R^(a))(R^(a)), or—O—P(═O)(OR^(a))₂ where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl,and each of these moieties can be optionally substituted as definedherein. In a non-limiting embodiment, a substituted alkyl can beselected from fluoromethyl, difluoromethyl, trifluoromethyl,2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl,3-hydroxypropyl, benzyl, and phenethyl.

As used herein, the term “alkoxy” refers to the group —O-alkyl,including from 1 to 10 carbon atoms (C₁₋₁₀) of a straight, branched,saturated cyclic configuration and combinations thereof, attached to theparent molecular structure through an oxygen. Examples include methoxy,ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, pentoxy, cyclopropyloxy,cyclohexyloxy and the like. “Lower alkoxy” refers to alkoxy groupscontaining one to six carbons. In some embodiments, C₁₋₃ alkoxy is analkoxy group that encompasses both straight and branched chain alkyls offrom 1 to 3 carbon atoms. Unless stated otherwise in the specification,an alkoxy group can be optionally substituted by one or moresubstituents which independently include: acyl, alkyl, alkenyl, alkynyl,alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido,amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl,heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo,haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio,arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate,silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,—Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a),—C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or 2), —P(═O)(R^(a))(R^(a)), or—O—P(═O)(OR^(a))₂ where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl,and each of these moieties can be optionally substituted as definedherein.

As used herein, the terms “aromatic” or “aryl” refer to a radical with 6to 14 ring atoms (e.g., C₆₋₁₄ aromatic or C₆₋₁₄ aryl) which has at leastone ring having a conjugated pi electron system which is carbocyclic(e.g., phenyl, fluorenyl, and naphthyl). In some embodiments, the arylis a C₆₋₁₀ aryl group. For example, bivalent radicals formed fromsubstituted benzene derivatives and having the free valences at ringatoms are named as substituted phenylene radicals. In other embodiments,bivalent radicals derived from univalent polycyclic hydrocarbon radicalswhose names end in“-yl” by removal of one hydrogen atom from the carbonatom with the free valence are named by adding “-idene” to the name ofthe corresponding univalent radical, e.g., a naphthyl group with twopoints of attachment is termed naphthylidene. Whenever it appearsherein, a numerical range such as “6 to 14 aryl” refers to each integerin the given range; e.g., “6 to 14 ring atoms” means that the aryl groupcan consist of 6 ring atoms, 7 ring atoms, etc., up to and including 14ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of ring atoms) groups. Polycyclic arylgroups include bicycles, tricycles, tetracycles, and the like. In amulti-ring group, only one ring is required to be aromatic, so groupssuch as indanyl are encompassed by the aryl definition. Non-limitingexamples of aryl groups include phenyl, phenalenyl, naphthalenyl,tetrahydronaphthyl, phenanthrenyl, anthracenyl, fluorenyl, indolyl,indanyl, and the like. Unless stated otherwise in the specification, anaryl moiety can be optionally substituted by one or more substituentswhich independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy,alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino,imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy,haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio,thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl,sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,—Si(R^(a))₃—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R_(a),—C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or 2), —P(═O)(R^(a))(R^(a)), or—O—P(═O)(OR^(a))₂ where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl,and each of these moieties can be optionally substituted as definedherein.

As used herein, the terms “cycloalkyl” and “carbocyclyl” each refers toa monocyclic or polycyclic radical that contains only carbon andhydrogen, and can be saturated or partially unsaturated. Partiallyunsaturated cycloalkyl groups can be termed “cycloalkenyl” if thecarbocycle contains at least one double bond, or “cycloalkynyl” if thecarbocycle contains at least one triple bond. Cycloalkyl groups includegroups having from 3 to 13 ring atoms (i.e., C₃₋₁₃ cycloalkyl). Wheneverit appears herein, a numerical range such as “3 to 10” refers to eachinteger in the given range; e.g., “3 to 13 carbon atoms” means that thecycloalkyl group can consist of 3 carbon atoms, 4 carbon atoms, 5 carbonatoms, etc., up to and including 13 carbon atoms. The term “cycloalkyl”also includes bridged and spiro-fused cyclic structures containing noheteroatoms. The term also includes monocyclic or fused-ring polycyclic(i.e., rings which share adjacent pairs of ring atoms) groups.Polycyclic aryl groups include bicycles, tricycles, tetracycles, and thelike. In some embodiments, “cycloalkyl” can be a C₃₋₈ cycloalkylradical. In some embodiments, “cycloalkyl” can be a C₃₋₅ cycloalkylradical. Illustrative examples of cycloalkyl groups include, but are notlimited to the following moieties: C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclobutyl (C₄), cyclopentyl (C₅),cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl(C₆) and the like. Examples of C₃₋₇ carbocyclyl groups include norbornyl(C₇). Examples of C₃₋₈ carbocyclyl groups include the aforementionedC₃₋₇ carbocyclyl groups as well as cycloheptyl (C₇), cycloheptadienyl(C₇), cycloheptatrienyl (C₇), cyclooctyl (C₈), bicyclo[2.2.1]heptanyl,bicyclo[2.2.2]octanyl, and the like. Examples of C₃₋₁₃ carbocyclylgroups include the aforementioned C₃₋₈ carbocyclyl groups as well asoctahydro-1H indenyl, decahydronaphthalenyl, spiro[4.5]decanyl and thelike. Unless stated otherwise in the specification, a cycloalkyl groupcan be optionally substituted by one or more substituents whichindependently include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl,cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino, azide,carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl,heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy,haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio,thiocarbonyl, nitro, oxo, phosphate, phosphonate, phosphinate, silyl,sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,—Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a),—C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂, —N(R^(a))C(NR^(a))N(R^(a))₂,—N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or 2), —P(═O)(R^(a))(R^(a)), or—O—P(═O)(OR^(a))₂ where each R^(a) is independently hydrogen, alkyl,haloalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl,heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl,and each of these moieties can be optionally substituted as definedherein. The terms “cycloalkenyl” and “cycloalkynyl” mirror the abovedescription of “cycloalkyl” wherein the prefix “alk” is replaced with“alken” or “alkyn” respectively, and the parent “alkenyl” or “alkynyl”terms are as described herein. For example, a cycloalkenyl group canhave 3 to 13 ring atoms, such as 5 to 8 ring atoms. In some embodiments,a cycloalkynyl group can have 5 to 13 ring atoms.

As used herein, the term “halide”, “halo”, or, alternatively, “halogen”means fluoro, chioro, bromo or iodo. The terms “haloalkyl,”“haloalkenyl,” “haloalkynyl” and “haloalkoxy” include alkyl, alkenyl,alkynyl and alkoxy structures that are substituted with one or more halogroups or with combinations thereof. For example, the terms“fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxygroups, respectively, in which the halo is fluorine, such as, but notlimited to, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, and the like. Each of the alkyl, alkenyl,alkynyl and alkoxy groups are as defined herein and can be optionallyfurther substituted as defined herein.

As used herein, the term “heteroalkyl” refers to an alkyl radical, whichhave one or more skeletal chain atoms selected from an atom other thancarbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinationsthereof. A numerical range can be given, e.g., C₁₋₄ heteroalkyl whichrefers to the chain length in total, which in this example is 4 atomslong. For example, a —CH₂OCH₂CH₃ radical is referred to as a “C₄”heteroalkyl, which includes the heteroatom center in the atom chainlength description. Connection to the parent molecular structure can bethrough either a heteroatom or a carbon in the heteroalkyl chain. Forexample, an N-containing heteroalkyl moiety refers to a group in whichat least one of the skeletal atoms is a nitrogen atom. One or moreheteroatom(s) in the heteroalkyl radical can be optionally oxidized. Oneor more nitrogen atoms, if present, can also be optionally quaternized.For example, heteroalkyl also includes skeletal chains substituted withone or more nitrogen oxide (—O—) substituents. Exemplary heteroalkylgroups include, without limitation, ethers such as methoxyethanyl(—CH₂CH₂OCH₃), ethoxymethanyl (—CH₂OCH₂CH₃), (methoxymethoxy)ethanyl(—CH₂CH₂OCH₂OCH₃), (methoxymethoxy) methanyl (—CH₂OCH₂OCH₃) and(methoxyethoxy)methanyl (—CH₂OCH₂CH₂OCH₃) and the like; amines such as(—CH₂CH₂NHCH₃, —CH₂CH₂N(CH₃)₂, —CH₂NHCH₂CH₃, —CH₂N(CH₂CH₃)(CH₃)) and thelike.

As used herein, the term “heteroaryl” or, alternatively,“heteroaromatic” refers to a refers to a radical of a 5-18 memberedmonocyclic or polycyclic (e.g., bicyclic, tricyclic, tetracyclic and thelike) aromatic ring system (e.g., having 6, 10 or 14 π electrons sharedin a cyclic array) having ring carbon atoms and 1-6 ring heteroatomsprovided in the aromatic ring system, wherein each heteroatom isindependently selected from nitrogen, oxygen, phosphorous and sulfur(“5-18 membered heteroaryl”). Heteroaryl polycyclic ring systems caninclude one or more heteroatoms in one or both rings. Whenever itappears herein, a numerical range such as “5 to 18” refers to eachinteger in the given range; e.g., “5 to 18 ring atoms” means that theheteroaryl group can consist of 5 ring atoms, 6 ring atoms, etc., up toand including 18 ring atoms. In some instances, a heteroaryl can have 5to 14 ring atoms. In some embodiments, the heteroaryl has, for example,bivalent radicals derived from univalent heteroaryl radicals whose namesend in “-yl” by removal of one hydrogen atom from the atom with the freevalence are named by adding “-ene” to the name of the correspondingunivalent radical, e.g., a pyridyl group with two points of attachmentis a pyridylene.

For example, an N-containing “heteroaromatic” or “heteroaryl” moietyrefers to an aromatic group in which at least one of the skeletal atomsof the ring is a nitrogen atom. One or more heteroatom(s) in theheteroaryl radical can be optionally oxidized. One or more nitrogenatoms, if present, can also be optionally quaternized. Heteroaryl alsoincludes ring systems substituted with one or more nitrogen oxide (—O—)substituents, such as pyridinyl N-oxides. The heteroaryl is attached tothe parent molecular structure through any atom of the ring(s).

“Heteroaryl” also includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more aryl groups wherein the pointof attachment to the parent molecular structure is either on the aryl oron the heteroaryl ring, or wherein the heteroaryl ring, as definedabove, is fused with one or more cycloalkyl or heterocycyl groupswherein the point of attachment to the parent molecular structure is onthe heteroaryl ring. For polycyclic heteroaryl groups wherein one ringdoes not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl andthe like), the point of attachment to the parent molecular structure canbe on either ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl). In some embodiments, a heteroaryl group is a 5-10 memberedaromatic ring system having ring carbon atoms and 1-4 ring heteroatomsprovided in the aromatic ring system, wherein each heteroatom isindependently selected from nitrogen, oxygen, phosphorous, and sulfur(“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group isa 5-8 membered aromatic ring system having ring carbon atoms and 1-4ring heteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen, phosphorous,and sulfur (“5-8 membered heteroaryl”). In some embodiments, aheteroaryl group is a 5-6 membered aromatic ring system having ringcarbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, phosphorous, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, phosphorous, and sulfur. In someembodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatomsselected from nitrogen, oxygen, phosphorous, and sulfur. In someembodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selectedfrom nitrogen, oxygen, phosphorous, and sulfur.

Examples of heteroaryls include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl,benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, benzo[b][1,4] oxazinyl, 1,4-benzodioxanyl,benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl,benzopyranonyl, benzofurazanyl, benzothiazolyl, benzothienyl(benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl,5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5Hbenzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo [3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d] pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl,phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl,pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl,pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl,quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo [4,5] thieno[2,3-d]pyrimdinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl,thiazolyl, thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl,thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisein the specification, a heteroaryl moiety can be optionally substitutedby one or more substituents which independently include: acyl, alkyl,alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy,amino, amido, amidino, imino, azide, carbonate, carbamate, carbonyl,heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy,cyano, halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio,alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate,phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl,sulfonate, urea, —Si(R^(a))₃, —OR^(a), —SR^(a), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R_(a), —C(O)OR^(a), —OC(O)N(R^(a))₂, —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))C(O)N(R^(a))₂,—N(R^(a))C(NR^(a))N(R^(a))₂, —N(R^(a))S(O)_(t)N(R^(a))₂ (where t is 1 or2), —P(═O)(R^(a))(R^(a)), or —O—P(═O)(OR^(a))₂ where each R^(a) isindependently hydrogen, alkyl, haloalkyl, carbocyclyl, carbocyclylalkyl,aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl orheteroarylalkyl, and each of these moieties can be optionallysubstituted as defined herein.

As used herein, the term “antibody” refers to molecules that are capableof binding an epitope or antigenic determinant. The term is meant toinclude whole antibodies and antigen-binding fragments thereof,including single-chain antibodies. The antibodies can be from any animalorigin. Preferably, the antibodies are mammalian, e.g., human, murine,rabbit, goat, guinea pig, camel, horse and the like, or other suitableanimals. Antibodies may recognize polypeptide or polynucleotideantigens. The term includes active fragments, including for example, anantigen binding fragment of an immunoglobulin, a variable and/orconstant region of a heavy chain, a variable and/or constant region of alight chain, a complementarity determining region (cdr), and a frameworkregion. The terms include polyclonal and monoclonal antibodypreparations, as well as preparations including hybrid antibodies,altered antibodies, chimeric antibodies, hybrid antibody molecules,F(ab)₂ and F(ab) fragments; Fv molecules (for example, noncovalentheterodimers), dimeric and trimeric antibody fragment constructs;minibodies, humanized antibody molecules, and any functional fragmentsobtained from such molecules, wherein such fragments retain specificbinding.

As used herein, the term “epitope” refers to basic element or smallestunit of recognition by an individual antibody or T-cell receptor, andthus the particular domain, region or molecular structure to which saidantibody or T-cell receptor binds. An antigen may consist of numerousepitopes while a hapten, typically, may possess few epitopes.

As used herein, the term “immune system” diseases or conditions refersto a group of conditions characterized by a dysfunctioning immunesystem. These disorders can be characterized in several different ways:by the component(s) of the immune system affected, by whether the immunesystem is overactive or underactive, or by whether the condition iscongenital or acquired. Autoimmune diseases or conditions are amongimmune system diseases or conditions.

As used herein, the term “inflammatory” diseases or conditions refers toa group of conditions including, rheumatoid arthritis, osteoarthritis,juvenile idiopathic arthritis, psoriasis, allergic airway disease (e.g.,asthma, rhinitis), inflammatory bowel diseases (e.g., Crohn's disease,colitis), endotoxin-driven disease states (e.g., complications afterbypass surgery or chronic endotoxin states contributing to e.g. chroniccardiac failure), and related diseases involving cartilage, such as thatof the joints.

As used herein, the term “autoimmune” diseases or conditions refers toconditions arising from an abnormal immune response to a normal bodypart. Examples of include, but not limited to rheumatoid arthritis,lupus, Alzheimer's disease, multiple sclerosis, Parkinson's disease,inflammatory bowel disease, and psoriasis.

As used herein, the term “cancer” refers to or describes thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, sarcoma, blastoma and leukemia. More particularexamples of such cancers include squamous cell carcinoma, lung cancer,pancreatic cancer, cervical cancer, bladder cancer, hepatoma, breastcancer, colon carcinoma, and head and neck cancer.

As used herein, the term “tumor” refers to any malignant or neoplasticcell.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues, and arenot limited to a minimum length. Thus, peptides, oligopeptides, dimers,multimers, and the like, are included within the definition. Bothfull-length proteins and fragments thereof are encompassed by thedefinition. The terms also include post-expression modifications of thepolypeptide, for example, glycosylation, acetylation, phosphorylation,and the like. Furthermore, a “polypeptide” may refer to a protein whichincludes modifications, such as deletions, additions, and substitutions(generally conservative in nature), to the native sequence, as long asthe protein maintains the desired activity. These modifications may bedeliberate or may be accidental.

As used herein, the term “sample” refers to a sample from a human,animal, or to a research sample, e.g., a cell, tissue, organ, fluid,gas, aerosol, slurry, colloid, or coagulated material. The “sample” maybe tested in vivo, e.g., without removal from the human or animal, or itmay be tested in vitro. The sample may be tested after processing, e.g.,by histological methods. “Sample” also refers, e.g., to a cellcomprising a fluid or tissue sample or a cell separated from a fluid ortissue sample. “Sample” may also refer to a cell, tissue, organ, orfluid that is freshly taken from a human or animal, or to a cell,tissue, organ, or fluid that is processed or stored.

As used herein, the term an “isolated” or “substantially isolated”molecule (such as a polypeptide or polynucleotide) is one that has beenmanipulated to exist in a higher concentration than in nature or hasbeen removed from its native environment. For example, a subjectantibody is isolated, purified, substantially isolated, or substantiallypurified when at least 10%, or 20%, or 40%, or 50%, or 70%, or 90% ofnon-subject-antibody materials with which it is associated in naturehave been removed. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated.” Further, recombinant DNA moleculescontained in a vector are considered isolated for the purposes of thepresent invention. Isolated RNA molecules include in vivo or in vitroRNA replication products of DNA and RNA molecules. Isolated nucleic acidmolecules further include synthetically produced molecules.Additionally, vector molecules contained in recombinant host cells arealso isolated. Thus, not all “isolated” molecules need be “purified.”

As used herein, the term “purified” when used in reference to amolecule, it means that the concentration of the molecule being purifiedhas been increased relative to molecules associated with it in itsnatural environment, or environment in which it was produced, found orsynthesized. Naturally associated molecules include proteins, nucleicacids, lipids and sugars but generally do not include water, buffers,and reagents added to maintain the integrity or facilitate thepurification of the molecule being purified. According to thisdefinition, a substance may be 5% or more, 10% or more, 20% or more, 30%or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% ormore, 90% or more, 95% or more, 98% or more, 99% or more, or 100% purewhen considered relative to its contaminants.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel, orally available, potent and selectiveinhibitors or inactivators of PADs, pharmaceutical compositions andmethods of use thereof. The compounds and pharmaceutical compositions ofthe invention may be used to treat immune system disorders andinflammatory diseases and conditions (e.g., rheumatoid arthritis,lupus). Diseases and conditions that may benefit from treatment usingthe compounds and pharmaceutical compositions of the invention includeulcerative colitis, spinal cord injury, breast cancer, andatherosclerosis.

Certain benzimidazole-based PAD inhibitors or inactivators disclosedherein display improved metabolic stability, cell permeability and/orpotency. It is believed that certain compounds of the invention canaccess binding regions within PAD2 and PAD4 that had not been previouslyaccessed.

For example, certain compounds disclosed herein possess superior PAD2inhibition (k_(inact)/K_(I)>300,000 k_(inact)/K_(I)) and selectivity(˜100-fold selective for PAD2 vs PAD4). Certain compounds disclosedherein exhibit improved cell permeability (e.g., over a 10-fold increasein Clog P compared to the peptide-based inhibitor Cl-amidine).

The invention also provides novel potent and selective molecular probes(e.g., imaging probes) for PADs based on the inhibitors or inactivatorsof PADs disclosed herein in conjugation with detectable labels such asfluorescent dyes, and methods of use thereof. The molecular imagingprobes of the invention may be used to screen compounds for PADinhibition or inactivation.

Aberrantly upregulated protein citrullination is associated with avariety of autoimmune diseases (e.g., rheumatoid arthritis, multiplesclerosis, lupus, and ulcerative colitis), as well as certain cancers.Given these disease links, the protein arginine deiminases have garneredsignificant recent interest. (Jones, et al. 2009 Curr Opin Drug DiscovDevel 12, 616-627; Bicker, et al. 2013 Biopolymers 99, 155-163;Vossenaar, et al. 2003 Bioessays 25, 1106-1118.)

The most deeply investigated disease associated with aberrantlyincreased PAD activity is RA, where these patients produceautoantibodies that target numerous citrullinated proteins (e.g.,citrullinated keratin, fibrin, vimentin and enolase). Importantly, thepresence of these autoantibodies is the most specific diagnostic testavailable for RA. The presence of these anti-citrullinated proteinantibodies (i.e., ACPA) is highly predictive of both disease incidenceand severity. (Van Steendam, et al. 2011 Rheumatology (Oxford) 50,830-837; Puszczewicz, et al. 2011 Arch Med Sci 7, 189-194; van Boekel,et al. 2002 Arthritis Res 4, 87-93; Masson-Bessiere, et al. 2001JImmunol 166, 4177-4184; Burska, et al. 2014 Mediators Inflamm 2014,492873.)

In addition to ACPA, the PADs themselves are present in the synovialjoints of patients with RA where they remain active and citrullinateproteins within the joints that bind ACPA, thereby setting up a classicpositive feedback loop that recruits additional immune cells into thejoint, the release of additional PAD isozymes into the synovium andenhanced protein citrullination and consequent inflammation. (Burska, etal. 2014 Mediators Inflamm 2014, 492873; Damgaard, et al. 2014 ArthritisRes Ther 16, 498.)

Without wishing to be bound by the theories discussed herein, thespecific cells that release PAD isozymes into the joints of RA patientsare likely neutrophils. Neutrophils are the predominant white blood cellin humans and are generally the first responders to signs ofinfection/inflammation. Depending on the environmental cues, a subset ofthese cells will undergo a novel form of cell death known as NeutrophilExtracellular trap formation (NET) or NETosis. Neutrophils have longbeen known to be important players in the development and progression ofRA as they are the predominant cell type in the synovial fluid of RApatients. Enhanced NETosis, as is observed in RA, also results in theexposure of citrullinated autoantigens, which is key to the progressionof RA. (Khandpur, et al. 2013 Sci Transl Med 5, 178ra140; Li, et al.2010 J Exp Med 207, 1853-1862; Brinkmann, et al. 2004 Science 303,1532-1535; Ottonello, et al. 2002 Rheumatology (Oxford) 41, 1249-1260;Weissmann, et al. 1984 Inflammation 8 Suppl, 53-14.)

In regard to characteristic features of other inflammatory diseases,enhanced citrullination in the inflamed regions indicates that abberantNETosis may contribute to these diseases as well. In addition, since thePADs are histone-modifying enzymes that contribute to the epigeneticcontrol of gene expression, there is emerging evidence to suggest thatenhanced PAD activity promotes an inflamed state by altering theexpression and/or activity of key cytokines and chemokines.(Kawalkowska, et al. 2016 Sci Rep 6, 26430; Loos, et al. 2008 Blood 112,2648-2656; Proost, et al. 2008 J Exp Med 205, 2085-2097; Struyf, et al.2009 J Immunol 182, 666-674.)

The role the PADs play in these diseases is further highlighted by theefficacy of several PAD inhibitors in a variety of pre-clinical diseasemodels. Specifically, the first-generation irreversible inhibitorCl-amidine has demonstrated efficacy in animal models of rheumatoidarthritis, lupus, ulcerative colitis, spinal cord injury, breast cancer,and atherosclerosis. (Khandpur, et al. 2013 Sci Transl Med 5, 178ra140;Wang, et al. 2012 J Biol Chem 287, 25941-25953; Chumanevich, et al. 2011Am J Physiol Gastrointest Liver Physiol 300, G929-938; Lange, et al.2011 Dev Biol 355, 205-214; Causey, et al. 2011 J Med Chem 54,6919-6935; Knight, et al. 2012 Curr Opin Rheumatol 24, 441-450; Knight,et al. 2013 J Clin Invest 123, 2981-2993; Smith, et al. 2014 ArthritisRheumatol 66, 2532-2544; Knight, et al. 2014 Circ Res 114, 947-956;McElwee, et al. 2012 BMC Cancer 12, 500.)

The first-generation inhibitors suffer a number of limitations includingtheir susceptibility to proteolysis and poor membrane permeability. Thisprompted the development of second-generation inhibitors predicated onimproving metabolic stability and membrane permeability.

Reports have shown that a second-generation inhibitor, BB-Cl-amidine,exhibits enhanced efficacy over Cl-amidine in animal models of lupus andRA. Moreover, the allosteric inhibitor GSK199 also shows efficacy in anRA model. Together, these findings have validated the PADs as viabletherapeutic targets for a wide range of inflammatory conditions.(Kawalkowska, et al. 2016 Sci Rep 6, 26430; Knight, et al. 2015 AnnRheum Dis 74, 2199-2206; Ghari, et al. 2016 Sci Adv 2, e1501257; Willis,V. C. The Role of Citrullination in the Development of Mouse and HumanInflammatory Arthritis. University of Colorado Boulder, Boulder, Colo.,2012.)

The novel PAD inhibitors or inactivators disclosed herein may be used totreat a variety of diseases where PAD activity is dysregulated, forexample, RA, lupus, atherosclerosis as well as other inflammatorydiseases. These PAD inhibitors may also find use in treating individualswith spinal cord injuries as well as psoriasis. Specifically, certainPAD inhibitors disclosed herein exhibit enhanced potency and selectivityfor PAD2, and so they may be used to treat diseases where PAD2 activityis upregulated. Certain disclosed inhibitors may be useful in treatingcertain cancers such as HER2 overexpressing breast cancers and certainlung cancers, as well as in the treatment of multiple sclerosis.

The PAD molecular probes disclosed herein may be specifically designedwith a terminal alkyne so they can covalently modify the PAD enzyme andundergo a subsequent “click” reaction with either TAMRA-N₃ or Biotin-N₃.These probes may find use in identifying particular diseases where thePADs are upregulated, as well as identifying PAD isozyme-specificdiseases. These probes should also find utility in identifying proteinsthat interact with the PADs as well as in identifying compounds withinhibitory properties or binding affinities to PADs.

In one aspect, the invention generally relates to a compound having thestructural formula (I),

wherein

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof.

In certain embodiments, each of R_(a) and R_(b) is H.

In certain embodiments, L is a —(CH₂)_(n)—, wherein n is an integer from1 to about 4 (e.g., n is 1, 2, 3, or 4). In preferred certainembodiments, n is 3 and L is —(CH₂)₃—.

In certain embodiments, Y is N and Z is N—R₁ with the compound havingthe structural formula (II):

In certain embodiments, X is F. In certain embodiments, X is Cl.

In certain embodiments, R₁ is a C₁₋₆ alkyl group. In certainembodiments, R₁ is H.

In certain embodiments, each of R₂, R₃, R₄ and R₅ is H.

In certain embodiments, at least one of R₂, R₃, R₄ and R₅ is a groupselected from the group consisting of hydroxyl, F, Cl, C₁₋₆ alkyl, C₁₋₆alkoxy, alkynyl, CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F oralkyl.

In certain embodiments, one of R₂ and R₃ is a group selected from thegroup consisting of hydroxyl, F, Cl, C₁₋₆ alkyl, C₁₋₆ alkoxy, alkynyl,CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F or alkyl.

In certain embodiments, R₆ is a cyclic, nonaromatic, hydrocarbyl group.

In certain embodiments, R₆ is a cyclic, aromatic, hydrocarbyl group.

In certain embodiments, R₆ is a heterocyclic, nonaromatic, hydrocarbylgroup.

In certain embodiments, R₆ is a heterocyclic, aromatic, hydrocarbylgroup.

In certain embodiments, R₆ is selected from the group consisting of:

wherein

R₇ is independently selected from the group consisting of H, C₁₋₆ alkyl,C₁₋₆ alkoxy, hydroxyl, halogen atoms, aryl, —COOR₈, ethynyl, alkynyl,CF₂R_(c), and OCF₂R_(c), where RC is H, F or alkyl;

R₈ is independently selected from the group consisting of H and C₁₋₆alkyl; and

R₉ is CH₂, O, NH₂ or S.

In certain embodiments, R₇ is H.

In certain embodiments, R₈ is a C₁₋₆ alkyl group.

In another aspect, the invention generally relates to a pharmaceuticalcomposition comprising a compound having the structural formula of (I):

wherein,

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof, effective to treat,prevent, or reduce one or more diseases or disorders, in a mammal,including a human, and a pharmaceutically acceptable excipient, carrier,or diluent.

In certain embodiments, the one or more diseases or conditions areselected from the group consisting of immune system diseases ordisorders, inflammatory diseases or disorders, and cancer or relateddiseases or disorders.

In certain embodiments, the one or more diseases or conditions areselected from autoimmune diseases or disorders.

In certain embodiments, the one or more diseases or conditions areselected from the group consisting of lupus, rheumatoid arthritis,Alzheimer's disease, multiple sclerosis and Parkinson's disease.

In certain embodiments, the disease or condition is lupus.

In certain embodiments, the disease or condition is rheumatoidarthritis.

Pharmaceutical compositions of the invention includes that of a compoundof the invention disclosed herein.

In yet another aspect, the invention generally relates to a unit dosageform comprising a pharmaceutical composition disclosed herein.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition comprising a compound having the structural formula of (I):

wherein,

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c), and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof, and a pharmaceuticallyacceptable excipient, carrier, or diluent, effective to treat, prevent,or reduce one or more diseases or disorders, in a mammal, including ahuman.

In certain embodiments, the one or more diseases or conditions areselected from the group consisting of immune system diseases ordisorders, inflammatory diseases or disorders, and cancer or relateddiseases or disorders.

In certain embodiments, the one or more diseases or conditions areselected from autoimmune diseases or disorders

In certain embodiments, the one or more diseases or conditions areselected from the group consisting of lupus, rheumatoid arthritis,Alzheimer's disease, multiple sclerosis and Parkinson's disease.

In certain embodiments, the disease or condition is lupus.

In certain embodiments, the disease or condition is rheumatoidarthritis.

In yet another aspect, the invention generally relates to a method forinhibiting or inactivating a protein arginine deiminase, comprisingadministering to a subject in need thereof a pharmaceutical compositioncomprising a compound having the structural formula of (I):

wherein,

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c) and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety,

or a pharmaceutically acceptable form thereof, and a pharmaceuticallyacceptable excipient, carrier, or diluent, effective to inhibit orinactivate a biological function of a protein arginine deiminase, in amammal, including a human.

In certain embodiments, the protein arginine deiminase is selected fromthe group consisting of: PAD1, PAD2, PAD3 and PAD4.

In certain embodiments, the protein arginine deiminase is PAD2.

In certain embodiments, the protein arginine deiminase is PAD4.

In yet another aspect, the invention generally relates to a molecularimaging probe having the structural formula:

A_(F)-L_(F)-W   (II)

wherein

A_(F) is a group comprising an optically detectable moiety;

L_(F) is a linking group; and

W is group comprising a benzimidazole moiety, or a derivative or analogthereof, capable of inhibiting or inactivating a biological function ofa protein arginine deiminase.

In certain embodiments, A_(F) is a group comprising a fluorophore.

In certain embodiments, the fluorophore is selected from the groupconsisting of xanthene dyes, cyanine dyes, coumarin dyes and bodipydyes.

In certain embodiments, the fluorophore is a xanthene dye selected fromthe group consisting of fluorescein, eosins, and rhodamines.

In certain embodiments, the fluorophore is a cyanine dye.

In certain embodiments, the fluorophore is a coumarin dye.

In certain embodiments, the fluorophore is a bodipy dye.

In certain embodiments, the protein arginine deiminase is selected fromthe group consisting of: PAD1, PAD2, PAD3 and PAD4.

In certain embodiments, the protein arginine deiminase is PAD2.

In certain embodiments, the protein arginine deiminase is PAD4.

In certain embodiments, W is a monovalent radical derived from acompound having the structural formula:

wherein,

each of R_(a) and R_(b) is independently selected from the groupconsisting of H, D and F;

L is a bivalent hydrocarbyl linker, optionally with one or more carbonatoms replaced by a heteroatom selected from the group consisting of O,S and N;

X is a halogen atom;

Y is N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds;

Z is N—R₁, O or S;

R₁ is selected from the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃alkoxy, CF₃, COCH₃, and COCF₃ groups;

each of R₂, R₃, R₄ and R₅ is independently selected from the groupconsisting of: H, hydroxyl, halogen atom, C₁₋₆ alkyl, C₁₋₆ alkoxy,alkynyl, CF₂R_(c) and OCF₂R_(c) groups, where R_(c) is H, F or alkyl;and

R₆ is a group comprising a cyclic alkyl or aryl moiety.

In yet another aspect, the invention generally relates to a method foridentifying a protein arginine deiminase inhibitor or inactivator. Themethod includes: performing a competitive assay wherein a test compoundcompetes with a molecular imaging probe disclosed herein to bind to aprotein arginine deiminase; and measuring fluorescence to determine anamount of fluorescent protein arginine deiminase present in the testassay.

In certain embodiments, the method further includes: performing acontrol assay wherein the molecular imaging probe binds to the proteinarginine deiminase; and measuring fluorescence to determine an amount offluorescent protein arginine deiminase present in the control assay.

In certain embodiments, a change in fluorescence in the assay greaterthan a pre-selected value when compared to the control assay isindicative that the test compound is an inhibitor to the proteinarginine deiminase.

In certain embodiments, the change in fluorescence in the assay is adecrease in fluorescence in the assay.

EXAMPLES

TABLE 1a k_(inact)/K_(I) values for compounds 5a-w PAD1 k_(inact)/K_(I)PAD2 k_(inact)/K_(I) PAD3 k_(inact)/K_(I) PAD4 k_(inact)/K_(I) Compound(M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) 5a (X = F) 4500 (±300)^(a)475 (±20)^(a) 900 (±200)^(a) 8500 (±1300)^(a) 5b (X = Cl) 7950(±675)^(a) 500 (±50)^(a) 9770 (±350)^(a) 15250 (±675)^(a) 5c (X = F)1500 (±400)^(a) 785 (±60)^(a) 2800 (±500)^(a) 6500 (±700)^(a) 5d (X =Cl) 2800 (±550)^(a) 4275 (±250)^(a) 9875 (±500)^(a) 10000 (±1000)^(a) 5e(R₁ = R₂ = R₃ = R₄ = R₅ = H, 900 (±100)^(a) 1200 (±30)^(a) 3400(±140)^(a) 3750 (±250)^(a) X = F) 5f (R₁ = R₂ = R₃ = R₄ = R₅ = H, 16100(±1500)^(a) 4100 (±400)^(a) 6800 (±200)^(a) 13300 (±3000)^(a) X = Cl) 5g(R₁ = Me, 1280 (±120)^(a) 820 (±90)^(b) 1600 (±210)^(a) 450 (±50)^(b) R₂= R₃ = R₄ = R₅ = H, X = F) 5h (R₁ = Me, R₂ = H, 6750 (±500)^(a) 2430(±400)^(a) 6020 (±650)^(a) 3110 (±420)^(a) R₂ = R₃ = R₄ = R₅ = H, X =Cl) 5i (R₁ = Et, R₂ = H, 760 (±100)^(a) 1160 (±100)^(b) 1800 (±210)^(a)30 (±5)^(b) R₂ = R₃ = R₄ = R₅ = H, X = F) 5j (R₁ = Et, R₂ = H, 5060(±650)^(a) 4850 (±320)^(a) 17040 (±2100)^(a) 4150 (±420)^(a) R₂ = R₃ =R₄ = R₅ = H, X = Cl) 5k (R₁ = ^(i)Pr, R₂ = H, 20 (±10)^(a) 680 (±50)^(b)1700 (±190)^(a) 20 (±5)^(b) R₂ = R₃ = R₄ = R₅ = H, X = F) 5l (R₁ =^(i)Pr, R₂ = H, 1220 (±190)^(a) 1490 (±180)^(a) 11500 (±1500)^(a) 950(±130)^(a) R₂ = R₃ = R₄ = R₅ = H, X = Cl) 5m (R₁ = Me, 1870 (±180)^(a)7920 (±1100)^(a) 2900 (±310)^(a) 790 (±95)^(a) R₂ = R₃ = R₄ = R₅ = H, R₂= OMe, X = F) 5n (R₁ = Me, 34800 (±4200)^(a) 27800 (±3500)^(a) 5500(±810)^(a) 7900 (±710)^(a) R₂ = R₃ = R₄ = R₅ = H, R₂ = OMe, X = Cl) 5o(R₁ = R₂ = R₃ = R₄ = H, 1400 (±120)^(a) 1700 (±210)^(a) 1620 (±190)^(a)3200 (±380)^(a) R₅ = F, X = F) 5p (R₁ = R₂ = R₃ = R₄ = H, 20600(±2400)^(a) 5500 (±620)^(a) 10530 (±1100)^(a) 16800 (±1800)^(a) R₅ = F,X = Cl) 5q (R₁ = R₂ = R₅ = H, 1860 (±190)^(a) 1570 (±200)^(a) 1530(±140)^(a) 3600 (±420)^(a) R₃ = R₄ = F, X = F) 5r (R₁ = R₂ = R₅ = H,27400 (±3100)^(a) 5600 (±610)^(a) 14360 (±1500)^(a) 20220 (±2150)^(a) R₃= R₄ = F, X = Cl) 5s (R₁ = R₂ = H, R₃ = R₄ = 1790 (±230)^(a) 1420(±170)^(a) 1100 (±160)^(a) 4040 (±480)^(a) R₅ = F, X = F) 5t (R₁ = R₂ =H, R₃ = R₄ = 30480 (±5350)^(a) 5730 (±590)^(a) 7180 (±790)^(a) 21250(±2500)^(a) R₅ = F, X = Cl) 5u (R₁ = R₂ = R₃ = R₄ = H, 4650 (±80)^(a)975 (±30)^(a) 1025 (±25)^(a) 1770 (±110)^(a) R₅ = CCH, X = F) 5v (R₁ =R₂ = R₃ = R₄ = H, 415 (±70)^(a) 875 (±70)^(a) 375 (±50)^(a) 1300(±100)^(a) R₅ = CCH, X = Cl) ^(a)A single k_(obs) was determined.^(b)k_(inact)/K_(I) was determined from a linear fit.

TABLE 1b Summary of isozyme selectivity for compounds 5a-w Fold FoldFold Fold PAD1 PAD2 PAD3 PAD4 Compound Selectivity SelectivitySelectivity Selectivity 5a (X = F) 9.5 1.0 1.9 18 5b (X = Cl) 16 1.0 2031 5c (X = F) 1.9 1.0 3.6 8.3 5d (X = Cl) 1.0 1.5 3.5 3.6 5e (R₁ = R₂ =R₃ = R₄ = 1.0 1.3 3.8 4.2 R₅ = H, X = F) 5f (R₁ = R₂ = R₃ = R₄ = 3.9 1.01.7 3.2 R₅ = H, X = Cl) 5g (R₁ = Me, R₂ = R₃ = 2.8 1.8 3.6 1.0 R₄ = R₅ =H, X = F) 5h (R₁ = Me, R₂ = H, 2.8 1.0 2.5 1.3 R₂ = R₃ = R₄ = R₅ = H, X= Cl) 5i (R₁ = Et, R₂ = H, 25 39 60 1.0 R₂ = R₃ = R₄ = R₅ = H, X = F) 5j(R₁ = Et, R₂ = H, 1.2 1.2 4.1 1.0 R₂ = R₃ = R₄ = R₅ = H, X = Cl) 5k (R₁= ^(i)Pr, R₂ = H, 1.0 34 85 1.0 R₂ = R₃ = R₄ = R₅ = H, X = F) 5l (R₁ =^(i)Pr, R₂ = H, 1.3 1.6 12 1.0 R₂ = R₃ = R₄ = R₅ = H, X = Cl) 5m (R₁ =Me, R₂ = R₃ = 2.4 10 3.7 1.0 R₄ = R₅ = H, R₂ = OMe, X = F) 5n (R₁ = Me,R₂ = R₃ = 6.3 5.1 1.0 1.4 R₄ = R₅ = H, R₂ = OMe, X = Cl) 5o (R₁ = R₂ =R₃ = R₄ = 1.0 1.2 1.2 2.3 H, R₅ = F, X = F) 5p (R₁ = R₂ = R₃ = R₄ = 3.71.0 1.9 3.1 H, R₅ = F, X = Cl) 5q (R₁ = R₂ = R₅ = H, 1.2 1.0 1.0 2.4 R₃= R₄ = F, X = F) 5r (R₁ = R₂ = R₅ = H, 4.9 1.0 2.6 3.6 R₃ = R₄ = F, X =Cl) 5s (R₁ = R₂ = H R₃ = 1.6 1.3 1.0 3.7 R₄ = R5 = F, X = F) 5t (R₁ = R₂= H, R₃ = 5.3 1.0 1.3 3.7 R₄ = R₅ = F, X = Cl) 5u (R₁ = R₂ = R₃ = R₄ =4.8 1.0 1.1 1.8 H, R₅ = CCH, X = F) 5v (R₁ = R₂ = R₃ = R₄ = 1.1 2.3 1.03.5 H, R₅ = CCH, X = Cl)

TABLE 2a k_(inact)/K_(I) values for compounds 6a-j PAD1 k_(inact)/K_(I)PAD2 k_(inact)/K_(I) PAD3 k_(inact)/K_(I) PAD4 k_(inact)/K_(I) Compound(M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) 6a (R₁ = R₂ = H, X = F)16500 (±2200)^(a) 1500 (±300)^(a) 6500 (±300)^(a) 24300 (±1800)^(a) 6b(R₁ = R₂ = H, X = Cl) 28500 (±2900)^(a) 3450 (±100)^(a) 21250(±2750)^(a) 31000 (±1550)^(a) 6c (R₁ = Me, R₂ = H, X = F) 32300(±4400)^(a) 11800 1030 (±150)^(a) 800 (±90)^(b) (±1090)^(b) 6d (R₁ = Me,R₂ = H, 32200 (±3540)^(a) 14400 7500 (±810)^(a) 7800 (±770)^(a) X = Cl)(±1230)^(a) 6e (R₁ = Et, R₂ = H, X = F) 39000 (±4600)^(a) 31600 1750(±180)^(a) 1010 (±170)^(b) (±3030)^(b) 6f (R₁ = Et, R₂ = H, X = Cl)47100 (±4600)^(a) 17400 11000 (±1300)^(a) 13700 (±2100)^(a) (±1500)^(a)6g (R₁ = ^(i)Pr, R₂ = H, X = F) 22700 (±2400)^(a) 24700 2010 (±190)^(a)460 (±80)^(a) (±2900)^(a) 6h (R₁ = ^(i)Pr, R₂ = H, 49000 (±5100)^(a)27100 2700 (±290)^(a) 720 (±90)^(a) X = Cl) (±2400)^(a) 6i (R₁ = Me, R₂= OMe, 37600 (±3900)^(a) 20400 6800 (±770)^(a) 6040 (±610)^(a) X = F)(±1900)^(a) 6j (R₁ = Me, R₂ = OMe, 67300 (±7100)^(a) 18500  9600(±1200)^(a) 14100 X = Cl) (±1900)^(a) (±1600)^(a) ^(a)A single k_(obs)was determined. ^(b)k_(inact)/K_(I) was determined from a linear fit.

TABLE 2b Summary of isozyme selectivity for compounds 6a-j Fold FoldFold Fold PAD1 PAD2 PAD3 PAD4 Compound Selectivity SelectivitySelectivity Selectivity 6a (R₁ = R₂ = H, 11 1.0 4.3 16 X = F) 6b (R₁ =R₂ = H, 8.3 1.0 6.2 9.0 X = Cl) 6c (R₁ = Me, R₂ = H, 40 15 1.3 1.0 X =F) 6d (R₁ = Me, R₂ = H, 4.3 1.9 1.0 1.0 X = Cl) 6e (R₁ = Et, R₂ = H, 3931 1.7 1.0 X = F) 6f (R₁ = Et, R₂ = H, 4.3 1.6 1.0 1.2 X = Cl) 6g (R₁ =^(i)Pr, R₂ = H, 49 54 4.4 1.0 X = F) 6h (R₁ = ^(i)Pr, R₂ = H, 68 38 3.81.0 X = Cl) 6i (R₁ = Me, R₂ = 6.2 3.4 1.1 1.0 OMe, X = F) 6j (R₁ = Me,R2 = 7.0 1.9 1.0 1.5 OMe, X = Cl)

TABLE 3a k_(inact)/K_(I) values for compounds 7a-v PAD1 k_(inact)/K_(I)PAD2 k_(inact)/K_(I) PAD3 k_(inact)/K_(I) PAD4 k_(inact)/K_(I) Compound(M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) 7a (R₁ = H, R₂ = H, R₃ = H, 77180 (±15000)^(a) 10600 (±3290)^(c) 3200 (±450)^(a) 33100 X = F)(±3350)^(b) 7b (R₁ = H, R₂ = H, R₃ = H, 180830 65400 (±9400)^(a) 2802058100 X = Cl) (±23500)^(a) (±3100)^(a) (±4600)^(a) 7c (R₁ = Me, R₂ = H,68670 (±9600)^(a) 24800 (±3200)^(a) 2800 (±310)^(a) 2910 (±270)^(a) R₃ =H, X = F) 7d (R₁ = Me, R₂ = H, 146000 72900 (±9350)^(a) 48500 23900 R₃ =H, X = Cl) (±22500)^(a) (±3750)^(a) (±2760)^(a) 7e (R₁ = Et, R₂ = H, R₃= H, 40500 (±6400)^(a) 61600 (±7750)^(d) 1100 (±210)^(a) 1900 (±270)^(b)X = F) 7f (R₁ = Et, R₂ = H, R₃ = H, 132000 (±2200)^(a) 60500 (±8160)^(a)22600 12600 X = Cl) (±3400)^(a) (±1800)^(a) 7g (R₁ = ^(i)Pr, R₂ = H,6220 (±790)^(a) 10300 (±1300)^(a) 510 (±75)^(a) 1230 (±160)^(b) R₃ = H,X = F) 7h (R₁ = ^(i)Pr, R₂ = H, 57600 (±7560)^(a) 16900 (±2200)^(a) 6700(±560)^(a) 2100 (±310)^(a) R₃ = H, X = Cl) 7i (R₁ = H, R₂ = OMe,  91600(±12300)^(a) 17500 (±2130)^(a) 8250 (±980)^(a) 2180 (±420)^(a) R₃ = H, X= F) 7j (R₁ = H, R₂ = OMe, 130830 48140 (±6300)^(a) 30120 4340(±620)^(a) R₃ = H, X = Cl) (±35600)^(a) (±3820)^(a) 7k (R₁ = Me, R₂ =OMe, 129100 210300 4430 (±510)^(a) 14300 R₃ = H, X = F) (±26300)^(a)(±58200)^(e) (±4800)^(a) 7l (R₁ = Me, R₂ = OMe, 77900 (±7500)^(a) 7780019900 25300 R₃ = H, X = Cl) (±10900)^(a) (±2120)^(a) (±2410)^(a) 7m (R₁= Me, R₂ = OEt, 64400 (±8320)^(a) 94450 1200 (±150)^(a)  990 (±110)^(a)R₃ = H, X = F) (±17700)^(f) 7n (R₁ = Me, R₂ = OEt, 58900 (±4500)^(a)71850 (±8320)^(a) 6470 (±680)^(a) 2410 (±320)^(a) R₃ = H, X = Cl) 7o (R₁= Me, R₂ = H, 124900 117300 1030 (±90)^(a) 1230 (±140)^(a) R₃ = OMe, X =F) (±33500)^(a) (±19500)^(g) 7p (R₁ = Me, R₂ = H, 132800 54490(±6230)^(a) 13100 5410 (±570)^(a) R3 = OMe, X = Cl) (±27800)^(a)(±1450)^(a) 7q (R₁ = Et, R₂ = OMe, 59900 (±6320)^(a) 50700 (±4350)^(a)1600 (±180)^(a) 1580 (±140)^(a) R₃ = H, X = F) 7r (R₁ = Et, R₂ = OMe,107200 39440 (±4200)^(a) 6180 (±710)^(a) 2130 (±210)^(a) R₃ = H, X = Cl)(±12300)^(a) 7s (R₁ = H, R₂ = H, 58410 (±4320)^(a) 24880 (±3120)^(a)4570 (±630)^(a) 15850 (±1890) R₃ = OMe, X = F) 7t (R₁ = H, R₂ = H,127700 60240 (±8210)^(a) 17450 37800 R₃ = OMe, X = Cl) (±45300)^(a)(±1560)^(a) (±4120)^(a) 7u (R₁ = Et, R₂ = H, 18930 (±1900)^(a) 39460(±3870)^(a) 1980 (±270)^(a)  790 (±160)^(a) R₃ = OMe, X = F) 7v (R₁ =Et, R₂ = H, 60530 (±8650)^(a) 46780 (±7410)^(a) 12830 2930 (±380)^(a) R₃= OMe, X = Cl) (±1430)^(a) ^(a)A single k_(obs) was determined.^(b)k_(inact)/K_(I) was determined from a linear fit. ^(c)k_(inact) =2.24 min⁻¹, K_(I) = 210 μM. ^(d)k_(inact) = 2.02 min⁻¹, K_(I) = 32.9 μM.^(e)k_(inact) = 3.47 min⁻¹, K_(I) = 16.5 μM. ^(f)k_(inact) = 5.21 min⁻¹,K_(I) = 55.2 μM. ^(g)k_(inact) = 2.42 min⁻¹, K_(I) = 20.7 μM.

TABLE 3b Summary of isozyme selectivity for compounds 7a-v Fold FoldFold Fold PAD1 PAD2 PAD3 PAD4 Compound Selectivity SelectivitySelectivity Selectivity 7a (R₁ = H, R₂ = H, 24 3.3 1.0 10 R₃ = H, X = F)7b (R₁ = H, R₂ = H, 6.5 2.3 1.0 2.1 R₃ = H, X = Cl) 7c (R₁ = Me, R₂ = H,25 8.9 1.0 1.0 R₃ = H, X = F) 7d (R₁ = Me, R₂ = H, 61 3.1 2.0 1.0 R₃ =H, X = Cl) 7e (R₁ = Et, R₂ = H, 37 56 1.0 1.7 R₃ = H, X = F) 7f (R₁ =Et, R₂ = H, 10 4.8 1.8 1.0 R₃ = H, X = Cl) 7g (R₁ = ^(i)Pr, R₂ = H, 1220 1.0 2.4 R₃ = H, X = F) 7h (R₁ = ^(i)Pr, R₂ = H, 27 8.0 3.2 1.0 R₃ =H, X = Cl) 7i (R₁ = H, R₂ = OMe, 42 8.0 3.8 1.0 R₃ = H, X = F) 7j (R₁ =H, R₂ = OMe, 30 11 6.9 1.0 R₃ = H, X = Cl) 7k (R₁ = Me, R₂ = 29 47 1.03.2 OMe, R₃ = H, X = F) 7l (R₁ = Me, R₂ = 3.9 3.9 1.0 1.3 OMe, R₃ = H, X= Cl) 7m (R₁ = Me, R₂ = 65 95 1.2 1.0 OEt, R₃ = H, X = F) 7n (R₁ = Me,R₂ = 24 30 2.7 1.0 OEt, R₃ = H, X = Cl) 7o (R₁ = Me, R₂ = H, 121 114 1.01.2 R₃ = OMe, X = F) 7p (R₁ = Me, R₂ = H, 25 10 2.4 1.0 R₃ = OMe, X =Cl) 7q (R₁ = Et, R₂ = 38 32 1.0 1.0 OMe, R₃ = H, X = F) 7r (R₁ = Et, R₂= 50 19 2.9 1.0 OMe, R₃ = H, X = Cl) 7s (R₁ = H, R₂ = H, 13 5.4 1.0 3.5R₃ = OMe, X = F) 7t (R₁ = H, R₂ = H, 7.3 3.5 1.0 2.2 R₃ = OMe, X = Cl)7u (R₁ = Et, R₂ = H, 24 50 2.5 1.0 R₃ = OMe, X = F) 7v (R₁ = Et, R₂ = H,21 16 4.4 1.0 R₃ = OMe, X = Cl)

TABLE 4a k_(inact)/K_(I) values for compounds 8a-b′ PAD1 k_(inact)/K_(I)PAD2 k_(inact)/K_(I) PAD3 k_(inact)/K_(I) PAD4 k_(inact)/K_(I) Compound(M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) 8a (R₁ = H, R₂ = H, R₃ = H,48400 (±5210)^(a) 14400 (±1320)^(a) 14900 (±1250)^(a) 14900 R₄ = Et, X =F) (±1510)^(a) 8b (R₁ = H, R₂ = H, R₃ = H, 151700 53300 (±6420)^(a)35000 (±2740)^(a) 48600 R₄ = Et, X = Cl) (±28900)^(a) (±4600)^(a) 8c (R₁= Me, R₂ = H, 18500 (±2230)^(a) 32760 (±2890)^(a) 5750 (±610)^(a) 3030(±280)^(a) R₃ = H, R₄ = Et, X = F) 8d (R₁ = Me, R₂ = H, 78910(±9480)^(a) 71350 (±5210)^(a) 3770 (±240)^(a) 4540 (±330)^(a) R₃ = H, R₄= Et, X = Cl) 8e (R₁ = Et, R₂ = H, R₃ = H, 24100 (±1980)^(a) 40610(±4050)^(a) 1250 (±130)^(a) 1240 (±110)^(a) R₄ = Et, X = F) 8f (R₁ = Et,R₂ = H, R₃ = H, 57100 (±6050)^(a) 45100 (±3030)^(a) 6870 (±790)^(a) 3250(±280)^(a) R₄ = Et, X = Cl) 8g (R₁ = ^(i)Pr, R₂ = H, 4700 (±420)^(a)10400 (±890)^(a) 1600 (±190)^(a) 470 (±50)^(a) R₃ = H, R₄ = Et, X = F)8h (R₁ = ^(i)Pr, R₂ = H, 26900 (±1960)^(a) 16600 (±1540)^(a) 4800(±520)^(a) 2430 (±270)^(a) R₃ = H, R₄ = Et, X = Cl) 8i (R₁ = H, R₂ =OMe, 80210 (±9130)^(a) 23790 (±2830)^(a)  9170 (±1030)^(a) 750 (±95)^(a)R₃ = H, R₄ = Et, X = F) 8j (R₁ = H, R₂ = OMe, 91900 (±5230)^(a) 43180(±5180)^(a) 18790 (±2050)^(a) 18450 R₃ = H, R₄ = Et, X = Cl) (±1750)^(a)8k (R₁ = Me, R₂ = OMe,  70760 (±18200)^(a) 365400 4320 (±520)^(a) 4300(±410)^(b) R₃ = H, R₄ = Et, X = F) (±84900)^(c) 8l (R₁ = Me, R₂ = OMe,57800 (±5550)^(a)  74660 (±12330)^(a) 14840 (±1620)^(a) 19400 R₃ = H, R₄= Et, X = Cl) (±2100)^(a) 8m (R₁ = Me, R₂ = OEt, 29800 (±3140)^(a) 85600(±7950)^(a) 3470 (±210)^(a) 3350 (±150)^(a) R₃ = H, R₄ = Et, X = F) 8n(R₁ = Me, R₂ = OEt, 18100 (±1930)^(a)  65170 (±13200)^(a) 8280(±910)^(a) 1510 (±80)^(a)  R₃ = H, R₄ = Et, X = Cl) 8o (R₁ = Me, R₂ = H,50500 (±6120)^(a) 133700 1070 (±120)^(a) 4100 (±330)^(a) R₃ = OMe, R₄ =Et, X = F) (±19800)^(d) 8p (R₁ = Me, R₂ = H,  71100 (±14200)^(a) 61500(±7800)^(a) 17750 (±1630)^(a) 1900 (±170)^(a) R₃ = OMe, R₄ = Et, X = Cl)8q (R₁ = Me, R₂ = OMe, 51470 (±9100)^(a)  93300 (±10760)^(a) 1600(±370)^(a) 4200 (±470)^(a) R₃ = H, R₄ = ^(i)Pr, X = F) 8r (R₁ = Me, R₂ =OMe, 43920 (±3300)^(a) 51200 (±8260)^(a) 14480 (±3140)^(a) 12400 R₃ = H,R₄ = ^(i)Pr, X = Cl) (±1520)^(a) 8s (R₁ = Me, R₂ = OMe, 45970(±7300)^(a)  94320 (±14860)^(a) 1220 (±280)^(a) 4500 (±920)^(a) R₃ = H,R₄ = cyclopropyl, X = F) 8t (R₁ = Me, R₂ = OMe, 40440 (±1750)^(a) 57500(±7500)^(a)  9480 (±1720)^(a) 8580 (±3320)^(a) R₃ = H, R₄ = cyclopropyl,X = Cl) 8u (R₁ = Et, R₂ = OMe, 39100 (±4210)^(a)  55980 (±11400)^(e)1400 (±130)^(a) 2800 (±190)^(a) R₃ = H, R₄ = Et, X = F) 8v (R₁ = Et, R₂= OMe, 54400 (±4180)^(a) 46570 (±3540)^(a) 5970 (±460)^(a) 1750(±220)^(a) R₃ = H, R₄ = Et, X = Cl) 8w (R₁ = H, R₂ = H,  9360(±1020)^(a) 13540 (±1890)^(a) 7630 (±810)^(a) 2140 (±190)^(a) R₃ = OMe,R₄ = Et, X = F) 8x (R₁ = H, R₂ = H, 30270 (±5660)^(a) 35400 (±4280)^(a)16000 (±1890)^(a) 3890 (±420)^(a) R₃ = OMe, R₄ = Et, X = Cl) 8y (R₁ =Et, R₂ = H, 15170 (±2130)^(a) 37780 (±4250)^(a) 1480 (±130)^(a)  540(±110)^(a) R₃ = OMe, R₄ = Et, X = F) 8z (R₁ = Et, R₂ = H, 30070(±3190)^(a) 38210 (±4310)^(a) 11850 (±1540)^(a) 1410 (±120)^(a) R₃ =OMe, R₄ = Et, X = Cl) 8a′ (R₁ = Me, R₂ = OMe, 59760 (±6130)^(a) 2127002010 (±180)^(a) 2300 (±410)^(b) R₃ = H, R₄ = Me, X = F) (±57650)^(f) 8b′(R₁ = Me, R₂ = OMe, 43150 (±5100)^(a) 69880 (±6430)^(a) 13650(±1410)^(a) 6340 (±820)^(a) R₃ = H, R₄ = Me, X = F) ^(a)A single k_(obs)was determined. ^(b)k_(inact)/K_(I) was determined from a linear fit.^(c)k_(inact) = 1.94 min⁻¹, K_(I) = 5.3 μM. ^(d)k_(inact) = 2.50 min⁻¹,K_(I) = 18.7 μM. ^(e)k_(inact) = 1.31 min⁻¹, K_(I) = 23.4 μM.^(f)k_(inact) = 2.73 min⁻¹, K_(I) = 12.8 μM.

TABLE 4b Summary of isozyme selectivity for compounds 8a-b′ Fold FoldFold Fold PAD1 PAD2 PAD3 PAD4 Compound Selectivity SelectivitySelectivity Selectivity 8a (R₁ = H, R₂ = H, 3.4 1.0 1.0 1.0 R₃ = H, R₄ =Et, X = F) 8b (R₁ = H, R₂ = H, 4.3 1.5 1.0 1.4 R₃ = H, R₄ = Et, X = Cl)8c (R₁ = Me, R₂ = H, 6.1 11 1.9 1.0 R₃ = H, R₄ = Et, X = F) 8d (R₁ = Me,R₂ = H, 21 19 1.0 1.2 R₃ = H, R₄ = Et, X = Cl) 8e (R₁ = Et, R₂ = H, 1933 1.0 1.0 R₃ = H, R₄ = Et, X = F) 8f (R₁ = Et, R₂ = H, 18 14 2.1 1.0 R₃= H, R₄ = Et, X = Cl) 8g (R₁ = ^(i)Pr, R₂ = H, 10 22 3.4 1.0 R₃ = H, R₄= Et, X = F) 8h (R₁ = ^(i)Pr, R₂ = H, 11 6.8 2.0 1.0 R₃ = H, R₄ = Et, X= Cl) 8i (R₁ = H, R₂ = OMe, 107 32 12 1.0 R₃ = H, R₄ = Et, X = F) 8j (R₁= H, R₂ = OMe, 5.0 2.3 1.0 1.0 R₃ = H, R₄ = Et, X = Cl) 8k (R₁ = Me, R₂= OMe, 16 85 1.0 1.0 R₃ = H, R₄ = Et, X = F) 8l (R₁ = Me, R₂ = OMe, 3.95.0 1.0 1.3 R₃ = H, R₄ = Et, X = Cl) 8m (R₁ = Me, R₂ = OEt, 8.9 26 1.01.0 R₃ = H, R₄ = Et, X = F) 8n (R₁ = Me, R₂ = OEt, 12 43 5.5 1.0 R₃ = H,R₄ = Et, X = Cl) 8o (R₁ = Me, R₂ = H, 47 125 1.0 3.8 R₃ = OMe, R₄ = Et,X = F) 8p (R₁ = Me, R₂ = H, 37 32 9.3 1.0 R₃ = OMe R₄ = Et, X = Cl) 8q(R₁ = Me, R₂ = OMe, 32 58 1.0 2.6 R₃ = H, R₄ = ^(i)Pr, X = F) 8r (R₁ =Me, R₂ = OMe, 3.5 4.1 1.2 1.0 R₃ = H, R₄ = ^(i)Pr, X = Cl) 8s (R₁ = Me,R₂ = OMe, 38 77 1.0 3.8 R₃ = H, R₄ = cyclopropyl, X = F) 8t (R₁ = Me, R₂= OMe, 4.7 6.7 1.1 1.0 R₃ = H, R₄ = cyclopropyl, X = Cl) 8u (R₁ = Et, R₂= OMe, 28 40 2.0 1.0 R₃ = H, R₄ = Et, X = F) 8v (R₁ = Et, R₂ = OMe, 3127 3.4 1.0 R₃ = H, R₄ = Et, X = Cl) 8w (R₁ = H, R₂ = H, 4.4 6.3 3.6 1.0R₃ = OMe, R₄ = Et, X = F) 8x (R₁ = H, R₂ = H, 7.8 9.1 4.1 1.0 R₃ = OMe,R₄ = Et, X = Cl) 8y (R₁ = Et, R₂ = H, 28 70 2.7 1.0 R₃ = OMe, R₄ = Et, X= F) 8z (R₁ = Et, R₂ = H, 21 27 8.4 1.0 R₃ = OMe, R₄ = Et, X = Cl) 8a′(R₁ = Me, R₂ = OMe, 30 106 1.0 1.1 R₃ = H, R₄ = Me, X = F) 8b′ (R₁ = Me,R₂ = OMe, 6.8 11 2.2 1.0 R₃ = H, R₄ = Me, X = F)

TABLE 5a k_(inact)/K_(I) values for compounds 9a-f PAD1 k_(inact)/K_(I)PAD2 k_(inact)/K_(I) PAD3 k_(inact)/K_(I) PAD4 k_(inact)/K_(I) Compound(M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) 9a (R = Me, X = F) 2470(±270)^(a) 2400 (±220)^(b) 1200 (±90)^(a)  2430 (±370)^(b) 9b (R = Me, X= Cl) 19500 3310 (±250)^(b) 5200 (±710)^(a) 3850 (±410)^(b) (±2200)^(a)9c (R = Et, X = F) 950 (±70)^(a) 6100 (±760)^(b) 3100 (±440)^(a) 440(±90)^(b) 9d (R = Et, X = Cl) 12030 9150 (±760)^(b) 4700 (±920)^(a) 1300(±220)^(b) (±1100)^(a) 9e (R = ^(i)Pr, X = F) 1420 (±210)^(a) 3010(±290)^(a) 2100 (±190)^(a) 160 (±80)^(a) 9f (R = ^(i)Pr, X = Cl) 3360(±520)^(a) 7150 (±990)^(a) 4800 (±520)^(a)  680 (±110)^(a) ^(a)A single/k_(obs) was determined. ^(b)k_(inact)/K_(I) was determined from alinear fit.

TABLE 5b Summary of isozyme selectivity for compounds 9a-f Fold FoldFold Fold PAD1 PAD2 PAD3 PAD4 Compound Selectivity SelectivitySelectivity Selectivity 9a (R = Me, X = F) 2.1 2.0 1.0 2.0 9b (R = Me, X= Cl) 5.9 1.0 1.6 1.2 9c (R = Et, X = F) 2.2 14 7.0 1.0 9d (R = Et, X =Cl) 9.3 7.0 3.6 1.0 9e (R = ^(i)Pr, X = F) 8.9 19 13 1.0 9f (R = ^(i)Pr,X = Cl) 4.9 11 7.1 1.0

TABLE 6a k_(inact)/K_(I) values for compounds 10a-h and 11a-h PAD1k_(inact)/K_(I) PAD2 k_(inact)/K_(I) PAD3 k_(inact)/K_(I) PAD4k_(inact)/K_(I) Compound (M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹) (M⁻¹min⁻¹)10a + 11a (R = H, X = F) 12600 (±1450)^(a) 10400 (±930)^(b) 20030 27300(±1980)^(a) (±2910)^(b) 10b + 11b (R = H, X = Cl) 57300 (±9800)^(a)34300 32800 41400 (±4200)^(b) (±2890)^(a) (±3800)^(b) 10c + 11c (R = Me,X = F) 28850 (±2760)^(a) 27700 29800 3250 (±670)^(a) (±3100)^(a)(±1980)^(a) 10d + 11d (R = Me, 65800 (±9500)^(a) 63800 44100 14400 X =Cl) (±9800)^(a) (±8200)^(a) (±4200)^(a) 10e + 11e (R = Et, X = F) 29900(±3400)^(a) 43300  9500 (±1020)^(a) 1930 (±180)^(a) (±3500)^(a) 10f +11f (R = Et, X = Cl) 71600 (±8700)^(a) 78300 30100 32090 (±6400)^(a)(±2870)^(a) (±3050)^(a) 10g + 11g (R = ^(i)Pr, X = F) 10050 (±1100)^(a)13600 10300 (±960)^(a)  380 (±50)^(b) (±1200)^(b) 10h + 11h (R = ^(i)Pr,57300 (±4070)^(a) 26900 7100 (±830)^(a) 3700 (±320)^(b) X = Cl)(∓2900)^(b) ^(a)A single k_(obs) was determined. ^(b)k_(inact)/K_(I) wasdetermined from a linear fit.

TABLE 6b Summary of isozyme selectivity for compounds 10a-h and 11a-hFold Fold Fold Fold PAD1 PAD2 PAD3 PAD4 Compound Selectivity SelectivitySelectivity Selectivity 10a + 11a (R = H, 1.2 1.0 1.9 2.6 X = F) 10b +11b (R = H, 1.7 1.0 1.0 1.3 X = Cl) 10c + 11c (R = Me, 8.9 8.5 9.2 1.0 X= F) 10d + 11d (R = Me, 4.6 4.4 3.1 1.0 X = Cl) 10e + 11e (R = Et, 15 224.9 1.0 X = F) 10f + 11f (R = Et, 2.4 2.6 1.0 1.1 X = Cl) 10g + 11g (R =^(i)Pr, 26 36 27 1.0 X = F) 10h + 11h (R = ^(i)Pr, 15 7.3 1.9 1.0 X =Cl)

FIG. 1 shows exemplary concentration dependent labeling of recombinantPAD2 with BB—F-Yne (5u) (FIG. 1A) and with BB—Cl-Yne (5v) (FIG. 1C) andthe limit of detection (LOD) of BB—F-Yne (5u) and of BB—Cl-Yne (5v) forPAD2, (FIG. 1B) and (FIG. 1D), respectively. Decreasing concentrationsof PAD2 treated with BB—F-Yne (5u) and “Clicked” with TAMRA-N₃.Decreasing concentrations of PAD2 were treated with BB—Cl-Yne (5v) and“Clicked” with TAMRA-N₃.

FIG. 2 shows exemplary in vitro labeling of the four active PAD isozymeswith BB—F-Yne (5u). FIG. 2A shows concentration dependent labeling ofrecombinant PAD1 with BB—F-Yne (5u). PAD1 was treated with increasingconcentrations of BB—F-Yne (5u) and then “Clicked” with TAMRA-N₃. FIG.2B shows concentration Concentration dependent labeling of recombinantPAD2 with BB—F-Yne (5u). PAD2 was treated with increasing concentrationsof BB—F-Yne (5u) and then “Clicked” with TAMRA-N₃. FIG. 2C showsconcentration dependent labeling of recombinant PAD3 with BB—F-Yne (5u).PAD3 was treated with increasing concentrations of BB—F-Yne (5u) andthen “Clicked” with TAMRA-N₃. FIG. 2D shows concentration dependentlabeling of recombinant PAD4 with BB—F-Yne (5u). PAD4 was treated withincreasing concentrations of BB—F-Yne (5u) and then “Clicked” withTAMRA-N₃.

FIG. 3 shows exemplary cellular labeling of PAD2 with BB—F-Yne (5u) inionophore stimulated HEK293T/PAD2 cells. FIG. 3A shows HEK293T/PAD2cells treated with increasing concentrations of BB—F-Yne (5u) for 1 h.The cells were then harvested and probe labeled proteins were taggedwith TAMRA-N₃ to facilitate visualization after SDS-PAGE. (B&C)HEK293T/PAD2 cells were treated with increasing concentrations ofBB—F-Yne (5u) for 1 h. The cells were then harvested and probe labeledproteins were tagged with Biotin-TEV-N₃. Biotin tagged proteins werethen isolated on streptavidin agarose and the eluted proteins wereprobed for PAD2 (FIG. 3B) or biotinylated proteins using streptavidin-RP(FIG. 3C).

FIG. 4 shows exemplary cellular labeling of PAD2 with BB—Cl-Yne (5v) inionophore stimulated HEK293T/PAD2 cells. FIG. 4A shows HEK293T/PAD2cells treated with increasing concentrations of BB—Cl-Yne (5v) for 1 h.The cells were then harvested and probe labeled proteins were taggedwith TAMRA-N₃ to facilitate visualization after SDS-PAGE. FIG. 4B-4Cshow HEK293T/PAD2 cells treated with increasing concentrations ofBB—Cl-Yne (5v) for 1 h. The cells were then harvested and probe labeledproteins were tagged with Biotin-TEV-N₃. Biotin tagged proteins werethen isolated on streptavidin agarose and the eluted proteins wereprobed for PAD2 (FIG. 4B) or biotinylated proteins using streptavidin-RP(FIG. 4C).

FIG. 5 shows exemplary cellular labeling of PAD4 with BB—F-Yne (5u).FIG. 5A shows HEK293T/PAD4 cells were treated with increasingconcentrations of BB—F-Yne (5u) for 1 h. The cells were then harvestedand probe labeled proteins were tagged with TAMRA-N₃ to facilitatevisualization after SDS-PAGE. FIG. 5B shows HEK293T/PAD4 cells treatedwith increasing concentrations of BB—F-Yne (5u) for 1 h. The cells werethen harvested and probe labeled proteins were tagged withBiotin-TEV-N₃. Biotin tagged proteins were then isolated on streptavidinagarose and the eluted proteins were probed for PAD2.

EXPERIMENTAL Chemistry

¹H NMR were recorded at 400 (Bruker DRX-400 with a H/C/P/F QNP gradientprobe) or 500 MHz (Bruker BioSpin 500 MHz Advance III Digital NMR)spectrometer and ¹³C NMR spectra were recorded at 100 or 125 MHz;chemical shifts are reported in δ (ppm) relative to the internalchloroform-d (CDCl₃, 7.26 ppm) or methanol-d (CD₃OD, 3.31 ppm). ESI(HRMS) were recorded with a Micromass Q-TQF I. The purity of allcompounds was determined to be >95% purity as determined by ¹H NMR and¹³C NMR spectra, unless otherwise noted. TLC was performed on glassbacked silica gel plates (Uniplate) with spots visualized by UV light.All solvents were reagent grade and, when necessary, were purified anddried by standard methods. Concentrations of solutions after reactionsand extractions involved the use of a rotary evaporator operating atreduced pressure.

Chemicals and Proteins

Dithiothreitol (DTT), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), ammonium iron (III) sulfate dodecahydrate,tris(2-carboxyethyl)phosphine (TCEP), and thiosemicarbazide wereacquired from Sigma-Aldrich. Diacetylmonooxime (DAMO),N-α-benzoyl-L-arginine ethyl ester (BAEE), and N-α-benzoyl-L-arginineamide (BAA) were obtained from Acros. Detailed synthetic procedures aredescribed below. PADs 1, 2, 3, and 4 were purified as reported. (Causey,et al. 2011 J Med Chem 54, 6919-6935; Knuckley, et al. 2010 Biochemistry49, 4852-4863.) HEK293T and HEK293T cells stably expressing human PAD2(HEK293T/PAD2) or PAD4 (HEK293T/PAD4) were cultured as previouslydescribed. (Lewallen, et al. 2014 ACS Chem Biol 9, 913-921.)Biotin-TEV-N₃ was synthesized as previously reported. (Weerapana, etal., 2007 Nat Protoc 2, 1414-1425.) TAMRA-N₃ was obtained fromLumiprobe.

Inactivation Kinetics

Inactivation kinetic parameters were determined by incubating PAD1, 2,or 4 (2.0 μM) or PAD3 (5.0 μM) in a pre-warmed (10 min; 37° C.)inactivation mixture (50 mM HEPES, 10 mM CaCl₂, and 2 mM DTT, pH 7.6,with a final volume of 60 μL) containing various concentrations ofinhibitor. Aliquots were removed at various time points and added to apre-warmed (10 min, 37° C.) reaction mixture (50 mM HEPES, 50 mM NaCl,10 mM CaCl₂, 2 mM DTT, and 10 mM BAEE or 10 mM BAA in the case of PAD3;pH 7.6). After 15 min, reactions were quenched in liquid nitrogen andcitrulline production quantified using the COLDER assay. (Kearney, etal. 2005 Biochemistry 44, 10570-10582; Knipp, et al. 2000 Anal Biochem286, 257-264.) Data were plotted as a function of time and fit to eq 1,

v=v _(o) e ^(−kt)  eq 1,

using GraFit version 5.0.11, where v is velocity, v_(o) is initialvelocity, k (or k_(obs)) is the pseudo-first order rate constant ofinactivation, and t is time. When saturation was reached upon plottingk_(obs) versus inactivator concentration, the data were fit to eq 2,

k _(obs) =k _(inact)[I]/(K _(I)+[I])  eq 2,

using GraFit version 5.0.11, where k_(inact) corresponds to the maximalrate of inactivation and K_(I) is the concentration of inhibitor thatgives half-maximal inactivation. If the plot of k_(obs) versus [I] waslinear and did not saturate, then the value for k_(inact)/K_(I) equaledthe slope of the line.In Vitro Labeling of PADs with “Clickable” Probes BB—F-Yne (5u),BB—Cl—Yne (5v) and BIFYne (5w)

Increasing concentrations of 5u-w (0 to 10 μM) were incubated withrecombinant PADs (1 μM) in the presence of CaCl₂ in 1×PBS (2 mM) at 37°C. for 1 h. The probe labeled enzymes were coupled to TAMRA-N₃ (20 μM)in the presence of 1×TBTA (0.31 mM), sodium ascorbate (2 mM) and freshlyprepared CuSO₄ (1 mM). The tubes were gently tumbled for 1 h. Thereactions were quenched with 6×SDS loading buffer and separated bySDS-PAGE (12.5% gel). The bands were visualized by scanning the gel in atyphoon scanner (approximate excitation/emission maxima ˜546/579,respectively).

In Vitro Labeling of PAD2 with BB—F-Yne (5u) and BB—Cl-Yne (5v) toDetermine their Limit of Detection (LOD)

BB—F-Yne (5u) or BB—Cl-Yne (5v) (10 μM) were incubated with decreasingconcentrations of recombinant PAD2 (1.0 to 0.025 μM) in the presence ofCaCl₂ in 1×PBS (2 mM) at 37° C. for 1 h. The probe labeled enzymes werecoupled to TAMRA-N₃ (20 μM) in the presence of 1×TBTA (0.31 mM), sodiumascorbate (2 mM) and freshly prepared CuSO₄ (1 mM). The tubes weregently tumbled for 1 h. The reactions were quenched with 6×SDS loadingbuffer and separated by SDS-PAGE (12.5% gel). The bands were visualizedby scanning the gel in a typhoon scanner (approximateexcitation/emission maxima ˜546/579, respectively).

Cellular Labeling of PAD2 with BB—F-Yne (5u) and BB—Cl-Yne (5v) inIonomycin Stimulated HEK293T/PAD2 Cells

HEK293T cells stably expressing human PAD2 (HEK293T PAD2) were cultured.Cells were grown to ˜80% confluence (8×10⁶ cells), trypsinized, andquenched trypsin activity with complete media. The cells were harvestedby centrifugation at 1000×g for 2 min and washed 4× with 1×PBS. Cellswere resuspended in PBS at 8×10⁶ cells/mL and 4×10⁵ cells were added to0.65 mL tubes for subsequent assays.

HEK293T PAD2 cells were treated with increasing concentrations ofBB—F-Yne (5u) or BB—Cl-Yne (5v) (0 to 50 μM) in the presence of CaCl₂ (2mM) at 37° C. After 20 min, ionomycin (10 μM) was added and the cellsincubated for 1 h before addition of Triton X-100 (1% final in PBS) andsonication at 4° C. for 1 h. Lysates were cleared by centrifugation at21,000×g for 15 min. The soluble protein fraction was isolated andquantified by the DC assay (Biorad). Lysates (2 μg/μL, 50 μL total) were“Clicked” with TAMRA-N₃ (20 μM), 1×TBTA (0.31 mM), sodium ascorbate (2mM) and freshly prepared CuSO₄ (1 mM). The tubes were gently tumbled for2 h. The reactions were quenched by the addition of 6×SDS loading bufferand separated by SDS-PAGE (12.5% gel). The bands were visualized byscanning the gel in a typhoon scanner (approximate excitation/emissionmaxima ˜546/579, respectively).

Lysates (2 μg/μL, 500 μL total) were also “Clicked” with Bio-TEV-N₃ (100μM), 1×TBTA (0.31 mM), sodium ascorbate (2 mM) and freshly preparedCuSO₄ (1 mM). The tubes were gently tumbled for 2 h. The cloudy solutionwas transferred to a microconcentrator (10 kDa molecular weight cutoff)and centrifuged at 10000×g for 5 min at 4° C. to remove the excessbiotin azide. The protein was then resolubilized in 1×PBS with 0.2% SDS.Resolubilized protein samples were incubated with 100 μLstreptavidin-agarose beads (Thermo Scientific) at 4° C. for 16 h. Thesolutions were then incubated at rt for 1 h. The beads were washed with0.2% SDS/PBS, PBS, and water. The beads were pelleted by centrifugation(1,600×g, 3 min) between washes. To the washed beads, 2×SDS loadingbuffer was added and heated to 95° C. heat block for 15 min. Theresolubilized protein was separated by SDS-PAGE (12.5% gel) andtransferred to PVDF membranes (Biorad) at 80 V for 60 min. The membraneswere analyzed for PAD2 and for biotinylated proteins.

1. General Procedure for Synthesis of Benzene-1,2-Diamine Intermediates

To a stirred solution of 1a-c (1.0 eq) in DMF was added NHR₃ (3.0 eq) ina sealed tube. The reaction mixture was then heated to 70° C. for 12 h,cooled, and diluted with water. The product was then filtered, washedwith water, dried under vacuum, and obtained in 78-89% yield. Thisproduct was then dissolved in a THF/EtOH/H₂O (3:1:1) solvent system, andcooled to 0° C. A solution of Na₂S₂O₄ in water was then added dropwiseto the stirred mixture. The reaction mixture was then warmed to rt andallowed to stir for 4 h. Upon completion, saturated NaHCO₃ solution wasadded followed by EtOAc extraction. The combined organic layers werethen washed with deionized water. The organic layer was then separated,dried over anhydrous Na₂SO₄, filtered, and concentrated to give desiredproducts 2a-d in 64-76% yield.

N¹-ethyl-3-methoxybenzene-1,2-diamine (2a)

¹H NMR (CDCl₃; 400 MHz): δ 6.78 (t, J=8.2 Hz, 1H), 6.41 (t, J=8.4 Hz,2H), 3.85 (s, 3H), 3.19-3.14 (m, 2H), 1.30 (t, J=7.3 Hz, 3H). ¹³C NMR(CDCl₃; 100 MHz): δ 148.3, 138.6, 122.6, 119.6, 117.1, 111.8, 106.0,105.3, 101.7, 55.8, 55.6, 43.6, 38.9, 15.1. LRMS m/z calculated forC₉H₁₄N₂O (M+H⁺) 167.1; found 167.2.

3-ethoxy-N¹-methylbenzene-1,2-diamine (2b)

¹H NMR (CDCl₃; 400 MHz): δ 6.78 (t, J=8.3 Hz, 1H), 6.43-6.37 (m, 2H),4.09-4.03 (m, 2), 2.87 (s, 3H), 1.42 (t, J=7.4 Hz, 3H). ¹³C NMR (CDCl₃;100 MHz): δ 147.4, 139.6, 122.9, 119.6, 104.4, 102.9, 64.1, 31.2, 15.1.LRMS m/z calculated for C₉H₁₄N₂O (M+H⁺) 167.1; found 167.2.

4-methoxy-N¹-methylbenzene-1,2-diamine (2c)

¹H NMR (CDCl₃; 400 MHz): δ 6.89 (dd, J=2.2 Hz, J=8.7 Hz, 1H), 6.52 (d,J=8.2 Hz, 1H), 6.27-6.24 (m, 1H), 3.79 (s, 3H), 3.66 (s, 3H). ¹³C NMR(CDCl₃; 100 MHz): δ 153.6, 143.6, 136.4, 132.1, 118.2, 113.2, 112.8,109.6, 103.8, 103.2, 102.2, 55.5, 31.8, 31.0, 29.3. LRMS m/z calculatedfor C₈H₁₂N₂O (M+H⁺) 152.1; found 152.2.

4-methoxy-N¹-ethylbenzene-1,2-diamine (2d)

¹H NMR (CDCl₃; 400 MHz): δ 7.10 (dd, J=2.3 Hz, J=8.2 Hz, 1H), 6.54 (d,J=8.2 Hz, 1H), 6.30-6.27 (m, 1H), 3.77 (s, 3H), 3.00 (m, 2H), 1.19 (t,3H). ¹³C NMR (CDCl₃; 100 MHz): δ 153.8, 137.1, 131.0, 118.3, 114.2,111.5, 109.1, 104.1, 103.8, 103.1, 102.8, 101.7, 55.9, 55.5, 39.7, 38.5,15.2, 14.9. LRMS m/z calculated for C₉H₁₄N₂O (M+H⁺) 167.1; found 167.1.

2. General Procedure for Synthesis of Benzimidazole Intermediates

To a stirred solution of Fmoc-Orn(Boc)-OH (3, 1.0 eq) in DMF was addedN¹R₁benzene-1,2-diamine (1.0 eq), DIPEA (3.0 eq), followed by HBTU (2.0eq) and HOBt, (2.0 eq). This reaction mixture was stirred at rt under N₂atmosphere for 12 h, and then diluted with water. The product wasfiltered, washed with water, and dried under vacuum, and obtained in81-92% yield. This product was dissolved in glacial AcOH/toluene (1:1)and heated at 80° C. for 16 h. The reaction mixture was evaporated todryness and diluted with water to precipitate the desired product(Fmoc-Orn(Boc)-benzimidazole) which was isolated by vacuum filtration in84-96% yield. This compound was then treated with 20% piperidine in DMFfor 20 min at rt to cleave the Fmoc group and the reaction was dilutedwith hexanes to remove the byproduct. The oily product was furtherpurified by column chromatography using MeOH and CH₂Cl₂ as the eluent togive 4a-j in 45-56% yield.

tert-butyl-(4-amino-4-(1H-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4a)

¹H NMR (CDCl₃; 400 MHz): δ 7.55-7.52 (m, 2H), 7.22-7.19 (m, 2H), 4.33(t, J=6.7 Hz, 1H), 3.31-3.22 (m, 1H), 3.11-3.02 (m, 1H), 2.01-1.92 (m,1H), 1.83-1.75 (m, 1H), 1.69-1.49 (m, 2H), 1.44 (s, 9H). ¹³C NMR (CDCl₃;100 MHz): δ 156.5, 122.1, 115.1, 79.5, 49.6, 39.5, 34.4, 28.3, 26.5.LRMS m/z calculated for C₁₆H₂₄N₄O₂ (M+H⁺) 305.2; found 305.1.

tert-butyl-(4-amino-4-(1-methyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4b)

¹H NMR (CDCl₃; 400 MHz): δ 7.73 (dd, J=2.6 Hz, 6.7 Hz, 1H), 7.33-7.29(m, 1H), 7.28-7.25 (m, 2H), 4.28 (t, J=6.8 Hz, 1H), 3.79 (s, 3H),3.18-3.13 (m, 2H), 2.10-2.01 (m, 1H), 1.94-1.83 (m, 1H), 1.71-1.53 (m,2H), 1.41 (s, 9H). ¹³C NMR (CDCl₃; 100 MHz): δ 149.6, 145.2, 139.2,127.0, 126.1, 122.6, 113.6, 100.0, 51.5, 43.8, 37.7, 33.7, 32.1, 29.8.LRMS m/z calculated for C₁₇H₂₆N₄O₂ (M+H⁺) 319.2; found 319.2.

tert-butyl-(4-amino-4-(1-ethyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4c)

¹H NMR (CDCl₃; 400 MHz): δ 7.67 (dd, J=2.2 Hz, J=7.2 Hz, 1H), 7.32-7.30(m, 1H), 7.24-7.19 (m, 2H), 4.27-4.22 (m, 1H), 4.21-4.12 (m, 2H),3.10-3.07 (m, 2H), 2.05-1.96 (m, 1H), 1.91-1.82 (m, 1H), 1.64-1.46 (m,2H), 1.37 (m, 12H). ¹³C NMR (CDCl₃; 100 MHz): δ 156.2, 155.9, 141.8,134.3, 122.7, 122.0, 119.3, 109.5, 79.0, 48.1, 39.9, 38.5, 34.2, 28.3,26.3, 15.1. LRMS m/z calculated for C₁₈H₂₈N₄O₂ (M+H⁺) 333.2; found333.3.

tert-butyl-(4-amino-4-(1-isopropyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4d)

¹H NMR (CDCl₃; 400 MHz): δ 7.71 (dd, J=2.4 Hz, 7.3 Hz, 1H), 7.52 (dd,J=2.1 Hz, J=6.3 Hz, 1H), 7.21-7.19 (m, 2H), 4.89-4.77 (m, 1H), 4.19 (t,J=6.8 Hz, 1H), 3.18-3.08 (m, 2H), 2.06-1.96 (m, 1H), 1.86-1.77 (m, 1H),1.65-1.62 (m, 6H), 1.60-1.52 (m, 2H), 1.40 (s, 9H). ¹³C NMR (CDCl₃; 100MHz): δ 156.7, 155.9, 142.8, 133.7, 121.9, 121.6, 119.5, 111.9, 78.8,49.0, 47.5, 40.3, 34.4, 28.2, 26.4, 21.3. LRMS m/z calculated forC₁₉H₃₀N₄O₂ (M+H⁺) 347.2; found 347.2.

tert-butyl-(4-amino-4-(5,6-difluoro-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4e)

¹H NMR (CDCl₃; 400 MHz): δ 7.28 (t, J=8.6 Hz, 2H), 4.38-4.35 (m, 1H),3.31-3.22 (m, 1H), 3.11-3.03 (m, 1H), 1.99-1.90 (m, 1H), 1.85-1.77 (m,1H), 1.68-1.58 (m, 1H), 1.56-1.49 (m, 1H), 1.43 (s, 9H). ¹³C NMR (CDCl₃;100 MHz): δ 156.8, 148.9, 147.0, 133.4, 102.5, 79.8, 49.5, 39.4, 33.7,28.4, 26.5. LRMS m/z calculated for C₁₆H₂₂F₂N₄O₂(M+H⁺) 341.2; found341.1.

tert-butyl-(4-amino-4-(4-methoxy-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4f)

¹H NMR (CDCl₃; 400 MHz): δ 7.08 (s, 2H), 6.58 (s, 1H), 4.58-4.53 (m,1H), 3.87 (s, 3H), 3.14-3.06 (m, 1H), 3.03-2.96 (m, 1H), 2.11-2.00 (m,1H), 1.97-1.90 (m, 1H), 1.51-1.39 (m, 2H), 1.36 (s, 9H). ¹³C NMR (CDCl₃;100 MHz): δ 177.1, 162.6, 156.5, 149.4, 138.2, 129.6, 123.6, 106.9,102.8, 79.4, 55.8, 55.5, 49.6, 39.5, 36.5, 32.6, 31.4, 28.3, 26.3, 22.5.LRMS m/z calculated for C₁₇H₂₆N₄O₃ (M+H⁺) 335.2; found 335.2.

tert-butyl-(4-amino-4-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4g)

¹H NMR (CDCl₃; 400 MHz): δ 7.19 (t, J=8.5 Hz, 1H), 6.92 (d, J=8.6 Hz,1H), 6.67 (d, J=8.3 Hz, 1H), 4.23-4.19 (m, 1H), 4.00 (s, 3H), 3.75 (s,3H), 3.17-3.11 (m, 2H), 2.05-1.96 (m, 1H), 1.92-1.82 (m, 1H), 1.70-1.53(m, 2H), 1.40 (s, 9H). ¹³C NMR (CDCl₃; 100 MHz): δ 156.2, 151.3, 137.3,132.0, 123.1, 102.5, 102.3, 79.2, 55.6, 48.5, 40.0, 34.1, 30.0, 28.2,26.5. LRMS m/z calculated for C₁₈H₂₈N₄O₃ (M+H⁺) 349.2; found 349.1.

tert-butyl-(4-amino-4-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4h)

¹H NMR (CDCl₃; 400 MHz): δ 7.15 (t, J=8.4 Hz, 1H), 6.92 (d, J=7.5 Hz,1H), 6.62 (d, J=8.4 Hz, 1H), 4.23-4.12 (m, 1H), 3.91 (s, 3H), 3.11-3.03(m, 2H), 2.01-1.92 (m, 1H), 1.88-1.79 (m, 1H), 1.61-1.44 (m, 2H), 1.38(t, J=7.5 Hz, 3H), 1.35 (s, 9H). ¹³C NMR (CDCl₃; 100 MHz): δ 156.1,155.2, 151.23, 135.9, 132.1, 123.2, 102.8, 79.0, 55.5, 50.5, 48.2, 39.9,38.4, 34.4, 28.2, 26.3, 15.1. LRMS m/z calculated for C₁₉H₃₀N₄O₃ (M+H⁺)363.2; found 363.3.

tert-butyl-(4-amino-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4i)

¹H NMR (CDCl₃; 400 MHz): δ 7.32 (d, J=8.4 Hz, 1H), 6.91 (s, 1H), 6.75(dd, J=2.3 Hz, J=8.6 Hz, 1H), 4.19 (m, 1H), 3.71 (s, 3H), 3.39 (s, 3H),1.91-1.83 (m, 1H), 1.76-1.69 (m, 1H), 1.48-1.42 (m, 2H), 1.34 (s, 9H).¹³C NMR (CDCl₃; 100 MHz): δ 156.7, 156.2, 138.2, 133.2, 115.8, 111.7,97.7, 79.2, 55.8, 50.3, 49.8, 39.7, 33.8, 28.5, 26.2. LRMS m/zcalculated for C₁₇H₂₆N₄O₃ (M+H⁺) 335.2; found 335.3.

tert-butyl-(4-amino-4-(5-methoxy-1-methyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4j)

¹H NMR (CDCl₃; 400 MHz): δ 7.21 (d, J=2.4 Hz, 1H), 7.17 (d, J=8.7 Hz,1H), 6.90 (dd, J=2.4 Hz, J=8.7 Hz, 1H), 4.20-4.18 (m, 1H), 3.82 (s, 3H),3.72 (s, 3H), 3.15-3.10 (m, 2H), 2.05-1.95 (m, 1H), 1.90-1.80 (m, 1H),1.68-1.49 (m, 2H), 1.40 (s, 9H). ¹³C NMR (CDCl₃; 100 MHz): δ 157.1,156.3, 155.9, 142.5, 130.4, 112.4, 109.7, 101.5, 79.0, 55.6, 48.3, 39.9,33.9, 29.9, 28.5, 26.6. LRMS m/z calculated for C₁₈H₂₈N₄O₃ (M+H⁺) 349.2;found 349.2.

tert-butyl-(4-amino-4-(5-methoxy-1-ethyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4k)

¹H NMR (CDCl₃; 400 MHz): δ 7.15-7.12 (m, 2H), 6.83 (dd, J=2.4 Hz, J=8.6Hz, 1H), 4.17-4.10 (m, 2H), 4.08-4.05 (m, 1H), 3.76 (s, 3H), 2.00-1.92(m, 1H), 1.84-1.77 (m, 1H), 1.61-1.53 (m, 1H), 1.50-1.43 (m, 1H), 1.34(s, 12H). ¹³C NMR (CDCl₃; 100 MHz): δ 156.1, 142.9, 129.2, 112.5, 109.9,101.8, 55.9, 50.3, 48.2, 40.1, 38.5, 34.5, 28.3, 26.5, 15.4. LRMS m/zcalculated for C₁₉H₃₀N₄O₃ (M+H⁺) 363.2; found 363.2.

tert-butyl-(4-amino-4-(4-ethoxy-1-methyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithine(4l)

¹H NMR (CDCl₃; 400 MHz): δ 7.16 (t, J=7.8 Hz, 1H), 6.89 (d, J=7.7 Hz,1H), 6.67 (d, J=7.8 Hz, 1H), 4.31-4.26 (m, 1H), 4.25-4.22 (m, 2H), 3.75(s, 3H), 3.16-3.11 (m, 2H), 2.04-1.95 (m, 1H), 1.90-1.82 (m, 1H),1.69-1.55 (m, 2H), 1.52 (t, J=7.2 Hz, 3H), 1.40 (s, 9H). ¹³C NMR (CDCl₃;100 MHz): δ 156.0, 150.2, 137.5, 132.4, 123.1, 103.7, 101.7, 79.1, 64.1,48.7, 39.8, 33.7, 30.2, 28.4, 26.5, 14.6. LRMS m/z calculated forC₁₉H₃₀N₄O₃ (M+H⁺) 363.2; found 363.1.

3a. General Procedure for Synthesis of Benzimidazole haloacetamidines5a-b.^(a)

To a stirred solution of 4a (1.0 eq) in THF/H₂O (1:1) was added TEA (3.0eq) followed by benzoyl chloride (1.0 eq) and allowed to stir at rt for3 h. Solvents were evaporated and the crude product was purified byreverse phase HPLC using MeCN:H₂O (0.5% TFA) as the eluent to give theproduct in 72-76% yield. This product was then treated with TFA toremove the Boc group giving the Bz-Orn-benzimidazole intermediate. Thesolvent was then evaporated to dryness and the crude material was driedin vacuo. To a stirred solution of the correspondingBz-Orn-benzimidazole intermediate in dry MeOH was added TEA (4.0 eq)followed by ethyl haloacetimidate HCl (2.0 eq). The reaction was stirredunder N₂ at rt for 3 h. Solvents were then evaporated under reducedpressure and the crude product was purified by reverse phase HPLC usingMeCN:H₂O (0.5% TFA) as an eluent to give compound 5a-b in 72-78% yield.

(N1-benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(5a)

¹H NMR (CD₃ OD; 400 MHz): δ 8.00-7.97 (m, 2H), 7.81-7.76 (m, 5H),5.66-5.63 (m, 1H), 5.30 (d, J=45.6 Hz, 2H), 3.55-3.50 (m, 2H), 2.39-2.34(m, 2H), 2.05-.192 (m, 1H), 1.90-1.83 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD)δ 170.5, 155.4, 134.1, 133.5, 133.3, 129.7, 128.8, 127.2, 115.2, 79.8,78.1, 42.8, 30.6, 25.2. HRMS m/z calculated for C₂₀H₂₃FN₅O (M+H⁺)368.1878; found 368.1880.

(N1-benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(5b)

¹H NMR (CD₃ OD; 400 MHz): δ 8.06-7.97 (m, 2H), 7.85-7.78 (m, 2H),7.62-7.59 (m, 3H), 7.58-7.50 (m, 2H), 5.67-5.63 (m, 1H), 4.39 (s, 2H),3.54-3.41 (m, 2H), 2.40-2.35 (m, 2H), 2.05-1.92 (m, 1H), 1.90-1.81 (m,1H). ¹³C NMR (100 MHz, CD₃ OD) δ 170.5, 164.5, 162.5, 155.3, 134.1,133.6, 133.0, 129.7, 128.8, 127.4, 115.1, 43.3, 40.1, 30.4, 25.0. HRMSm/z calculated for C₂₀H₂₃ClN₅O (M+H⁺) 384.1583; found 384.1588.

3b. General Procedure for Synthesis of Benzimidazole haloacetamidines5c-da

To a stirred solution of 4a (1.0 eq) in THF/H₂O (1:1) was added TEA (3.0eq) followed by naphthoyl chloride (1.0 eq) and allowed to stir at rtfor 3 h. Solvents were evaporated and the crude product was purified byreverse phase HPLC using MeCN:H₂O (0.5% TFA) as the eluent to give theproduct in 68-73% yield. This product was then treated with TFA toremove the Boc group giving the Naphthyl-Om-benzimidazole intermediate.The solvent was then evaporated to dryness and the crude material wasdried in vacuo. To a stirred solution of the correspondingNaphthyl-Orn-benzimidazole intermediate in dry MeOH was added TEA (4.0eq) followed by ethyl haloacetimidate HCl (2.0 eq). The reaction wasstirred under N₂ at rt for 3 h. Solvents were then evaporated underreduced pressure and the crude product was purified by reverse phaseHPLC using MeCN:H₂O (0.5% TFA) as an eluent to give compound 5c-d in30-40% yield.

(N1-[2-naphthoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(5c)

¹H NMR (CD₃ OD; 400 MHz): δ 8.58 (s, 1H), 8.11-7.93 (m, 4H), 7.81-7.74(m, 2H), 7.66-7.56 (m, 4H), 5.74-5.70 (m, 1H), 5.30 (d, J=45.6 Hz, 2H),3.58-3.50 (m, 2H), 2.44-2.40 (m, 2H), 2.10-1.86 (m, 2H). ¹³C NMR (100MHz, CD₃ OD) δ 170.5, 164.6, 164.4, 136.6, 133.9, 132.9, 131.3, 130.2,129.6, 129.5, 129.3, 128.8, 128.1, 127.4, 125.0, 115.1, 79.8, 78.1,42.8, 30.5, 25.2. HRMS m/z calculated for C₂₄H₂₅FN₅O (M+H⁺) 418.2035;found 418.2040.

(N1-[2-naphthoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(5d)

¹H NMR (CD₃ OD; 400 MHz): δ 8.58 (s, 1H), 8.12-7.92 (m, 4H), 7.81-7.73(m, 2H), 7.67-7.55 (m, 4H), 5.74-5.70 (m, 1H), 4.42 (s, 2H), 3.57-3.49(m, 2H), 2.43-2.39 (m, 2H), 2.10-1.85 (m, 2H). ¹³C NMR (100 MHz, CD₃ OD)δ 170.4, 169.4, 164.9, 163.0, 162.6, 154.6, 139.1, 133.8, 133.7, 131.7,131.2, 130.3, 128.9, 127.2, 115.2, 43.3, 40.1, 30.4, 25.0. HRMS m/zcalculated for C₂₄H₂₅ClN₅O (M+H⁺) 434.1739; found 434.1741.

3c. General Procedure for Synthesis of Benzimidazole haloacetamidines5e-w.^(a)

To a stirred solution of 4a-e, g (1.0 eq) in CH₂Cl₂ was added TEA (3.0eq) followed by biphenyl-4-carbonyl chloride (1.0 eq) and allowed tostir at rt for 3 h. Solvents were evaporated and the crude product waspurified by reverse phase HPLC using MeCN:H₂O (0.5% TFA) as the eluentto give the product in 69-80% yield. This product was then treated withTFA to remove the Boc group giving the 4-Ph-Bz-Orn-benzimidazoleintermediate. The solvent was then evaporated to dryness and the crudematerial was dried in vacuo. To a stirred solution of the corresponding4-Ph-Bz-Orn-benzimidazole intermediate in dry MeOH was added TEA (4.0eq) followed by ethyl haloacetimidate HCl (2.0 eq). The reaction wasstirred under N₂ at rt for 3 h. Solvents were then evaporated underreduced pressure and the crude product was purified by reverse phaseHPLC using MeCN:H₂O (0.5% TFA) as an eluent to give compound 5a-p in53-75% yield.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)5e)

¹H NMR (CD₃ OD; 400 MHz): δ 8.07 (d, J=8.4 Hz, 2H), 7.87-7.76 (m, 4H),7.77 (d, J=8.4 Hz, 2H), 7.69-7.58 (m, 2H), 7.51-7.47 (m, 2H), 7.43-7.39(m, 1H), 5.70-5.56 (m, 1H), 5.30 (d, J=45.2 Hz, 2H), 3.51-3.49 (m, 2H),2.42-2.36 (m, 2H), 2.06-1.83 (m, 2H). ¹³C NMR (100 MHz, CD₃ OD) δ 170.2,164.5, 155.4, 146.5, 141.0, 132.8, 132.7, 130.1, 129.5, 129.3, 128.2,127.5, 115.3, 79.8, 78.1, 42.8, 30.5, 25.2. HRMS m/z calculated forC₂₆H₂₇FN₅O (M+H⁺) 444.2191; found 444.2194.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-omithine)

¹H NMR (CD₃ OD; 400 MHz): δ 8.06 (d, J=8.4 Hz, 2H), 7.79 (dd, J=3.2 Hz,J=8.4 Hz, 2H), 7.74 (d, J=8.4 Hz, 2H), 7.67-7.64 (m, 2H), 7.58 (dd,J=3.2 Hz, J=8.4 Hz, 2H), 7.48-7.44 (m, 2H), 7.39-7.36 (m, 1H), 5.71-5.57(m, 1H), 4.40 (s, 2H), 3.56-3.43 (m, 2H), 2.47-2.33 (m, 2H), 2.09-2.01(m, 1H), 1.98-1.83 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 170.3, 164.8,155.4, 146.5, 141.0, 132.6, 132.5, 130.1, 129.5, 129.3, 128.2, 127.6,115.1, 43.3, 40.1 (2C), 30.6, 25.1. HRMS m/z calculated for C₂₆H₂₇ClN₅O(M+H⁺) 460.1896; found 460.1892.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(52)

¹H NMR (CD₃ OD; 400 MHz): δ 8.01 (d, J=8.4 Hz, 2H), 7.87 (dd, J=2.3 Hz,J=8.1 Hz, 1H), 7.81 (dd, J=2.3 Hz, J=7.2 Hz, 1H), 7.75 (d, J=8.3 Hz,2H), 7.66 (d, J=8.3 Hz, 2H), 7.63-7.58 (m, 2H), 7.47 (t, J=7.3 Hz, 2H),7.39 (t, J=7.8 Hz, 1H), 5.78-5.74 (m, 1H), 5.27 (d, J=45.4 Hz, 2H), 4.17(s, 3H), 3.54-3.44 (m, 2H), 2.45-2.26 (m, 2H), 2.06-2.01 (m, 1H),1.91-1.80 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 168.5, 163.2, 162.9,161.4, 160.9, 153.0, 145.1, 139.6, 133.6, 131.21, 128.7 (2C), 127.9,127.9, 126.8, 126.7, 125.5, 125.4, 115.2, 111.6, 78.5, 76.7, 45.7, 41.4,30.3, 28.9, 23.7. HRMS m/z calculated for C₂₇H₂₈FN₅O (M+H⁺) 458.2351;found 458.2349.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(5h)

¹H NMR (CD₃ OD; 400 MHz): δ 8.00 (d, J=8.3 Hz, 2H), 7.86 (dd, J=2.4 Hz,J=7.6 Hz, 1H), 7.80 (dd, J=2.4 Hz, J=7.2 Hz, 1H), 7.75 (d, J=8.7 Hz,2H), 7.68-7.65 (m, 2H), 7.62-7.57 (m, 2H), 7.47 (t, J=7.2 Hz, 2H), 7.39(t, J=7.2 Hz, 1H), 5.77-5.73 (m, 1H), 4.38 (s, 2H), 4.14 (s, 3H),3.53-3.41 (m, 2H), 2.46-2.29 (m, 2H), 2.08-1.97 (m, 1H), 1.93-1.81 (m,1H). ¹³C NMR (100 MHz, CD₃ OD) δ 168.5, 163.4, 161.3, 155.3, 152.9,145.1, 139.5, 134.9, 133.9, 133.7, 131.3, 128.7 (2C), 127.9, 127.9,126.8, 126.7, 125.3, 125.3, 115.3, 111.5, 45.7, 42.0, 38.7, 30.2, 29.0,23.5. HRMS m/z calculated for C₂₇H₂₈ClN₅O (M+H⁺) 474.2055; found474.2056.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-omithine)(5i)

¹H NMR (CD₃ OD; 400 MHz): δ 7.99 (d, J=8.6 Hz, 2H), 7.88 (dd, J=2.1 Hz,J=7.2 Hz, 1H), 7.81 (dd, J=2.2 Hz, J=7.1 Hz, 1H), 7.75 (d, J=8.6 Hz,2H), 7.66 (d, J=7.7 Hz, 2H), 7.61-7.56 (m, 2H), 7.47 (t, J=7.3 Hz, 2H),7.39 (t, J=7.7 Hz, 1H), 5.78-5.74 (m, 1H), 5.27 (d, J=45.2 Hz, 2H),4.66-4.58 (m, 2H), 3.54-3.45 (m, 2H), 2.45-2.36 (m, 1H), 2.34-2.25 (m,1H), 2.06-1.97 (m, 1H), 1.93-1.81 (m, 1H), 1.41 (t, J=7.1 Hz, 3H). ¹³CNMR (100 MHz, CD₃ OD) δ 168.2, 163.1, 163.0, 152.5, 145.1, 139.5, 132.8,132.3, 131.9, 131.4, 128.7 (2C), 127.9, 126.8, 126.7, 125.1, 124.9,123.2, 115.9, 111.6, 78.5, 76.7, 45.5, 41.6, 39.7, 29.4, 23.8, 13.6.HRMS m/z calculated for C₂₈H₃₀FN₅O (M+H) 472.2507; found 472.2506.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(5j)

¹H NMR (CD₃ OD; 400 MHz): δ 7.99 (d, J=8.3 Hz, 2H), 7.90 (dd, J=2.1 Hz,J=7.4 Hz, 1H), 7.81 (dd, J=2.4 Hz, J=7.3 Hz, 1H), 7.75 (d, J=8.3 Hz,2H), 7.66 (d, J=8.4 Hz, 2H), 7.62-7.56 (m, 2H), 7.46 (t, J=7.2 Hz, 2H),7.40 (t, J=7.3 Hz, 1H), 5.78-5.74 (m, 1H), 4.67-4.60, 4.37 (s, 2H),3.52-3.42 (m, 2H), 2.46-2.36 (m, 1H), 2.34-2.26 (m, 1H), 2.08-1.97 (m,1H), 1.93-1.82 (m, 1H), 1.54 (t, J=7.5 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 168.3, 163.4, 161.3, 161.0, 152.4, 145.1, 139.6, 134.6, 132.7,131.3, 128.7 (2C), 127.9, 126.8, 126.7, 125.2, 125.1, 115.7, 111.7,100.0, 56.1, 45.5, 42.1, 39.8, 38.7, 29.3, 23.7, 13.5. HRMS m/zcalculated for C₂₈H₃₀ClN₅O (M+H⁺) 488.2212; found 488.2214.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(5k)

¹H NMR (CD₃ OD; 400 MHz): δ 8.06-8.02 (m, 1H), 7.98 (d, J=8.5 Hz, 2H),7.83-7.79 (m, 1H), 7.75 (d, J=8.5 Hz, 2H), 7.66 (dd, J=2.1 Hz, J=8.1 Hz,2H), 7.57-7.53 (m, 2H), 7.47 (t, J=7.4 Hz, 2H), 7.39 (t, J=7.4 Hz, 1H),5.80-5.76 (m, 1H), 5.28 (d, J=45.3 Hz, 2H), 5.21-5.16 (m, 1H), 3.57-3.44(m, 2H), 2.45-2.36 (m, 1H), 2.35-2.27 (m, 1H), 2.06-1.96 (m, 1H),1.93-1.84 (m, 1H), 1.81 (d, J=6.6 Hz, 3H), 1.71 (d, J=6.6 Hz, 3H). ¹³CNMR (100 MHz, CD₃ OD) δ 168.4, 166.0, 165.1, 162.5, 161.4, 152.1, 145.1,139.5, 133.0, 131.1, 130.6, 128.7 (2C), 127.9, 126.8, 126.6, 125.7,125.4, 115.1, 114.4, 78.4, 76.7, 50.8, 46.0, 41.4, 29.0, 23.7, 19.6(2C). HRMS m/z calculated for C₂₉H₃₂FN₅O (M+H⁺) 486.2664; found486.2666.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(5l)

¹H NMR (CD₃ OD; 400 MHz): δ 8.05-8.03 (m, 1H), 7.99 (d, J=8.3 Hz, 2H),7.83-7.79 (m, 1H), 7.75 (d, J=8.4 Hz, 2H), 7.67 (dd, J=2.2 Hz, J=8.3 Hz,2H), 7.58-7.53 (m, 2H), 7.47 (t, J=7.3 Hz, 2H), 7.39 (t, J=7.6 Hz, 1H),5.80-5.76 (m, 1H), 5.25-5.15 (m, 1H), 4.38 (s, 2H), 3.55-3.42 (m, 2H),2.46-2.36 (m, 1H), 2.35-2.27 (m, 1H), 2.07-1.96 (m, 1H), 1.93-1.85 (m,1H), 1.81 (d, J=6.7 Hz, 3H), 1.72 (d, J=6.5 Hz, 3H). ¹³C NMR (100 MHz,CD₃ OD) δ 168.4, 166.2, 165.3, 163.3, 160.9, 152.1, 145.1, 139.5, 131.0,130.4, 128.6 (2C), 127.9, 126.8, 126.7, 125.9, 125.5, 115.6, 115.0,114.5, 52.8, 50.9, 46.1, 41.9, 38.6, 29.0, 23.6, 19.6 (2C). HRMS m/zcalculated for C₂₉H₃₂ClN₅O (M+H⁺) 502.2368; found 502.2370.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-omithine)(5m)

¹H NMR (CD₃ OD; 400 MHz): δ 7.97 (d, J=8.5 Hz, 2H), 7.75 (d, J=8.6 Hz,2H), 7.66 (dd, J=1.8 Hz, J=7.2 Hz, 2H), 7.49-7.37 (m, 4H), 7.28 (d,J=8.4 Hz, 2H), 6.99 (d, J=8.1 Hz, 2H), 5.69-5.64 (m, 1H), 5.27 (d,J=45.1 Hz, 2H), 4.03 (s, 3H), 4.01 (s, 3H), 3.51-3.41 (m, 2H), 2.34-2.28(m, 2H), 2.00-1.89 (m, 1H), 1.86-1.75 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD)δ 168.5, 163.0, 162.9, 161.3, 160.9, 152.1, 148.1, 144.9, 139.5, 134.6,131.0, 128.6, 127.9, 126.8, 126.7, 122.7, 117.9, 115.0, 105.9, 103.6,78.3, 76.6, 55.1, 46.0, 41.4, 30.7, 28.7, 23.5. HRMS m/z calculated forC₂₈H₃₀FN₅O₂(M+H⁺) 488.2456; found 488.2455.

(N1-[1,4′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(5n)

¹H NMR (CD₃ OD; 400 MHz): δ 7.98 (d, J=8.5 Hz, 2H), 7.74 (d, J=8.6 Hz,2H), 7.65 (dd, J=1.8 Hz, J=8.4 Hz, 2H), 7.48-7.36 (m, 4H), 7.29 (d,J=8.4 Hz, 2H), 7.00 (d, J=8.2 Hz, 2H), 5.69-5.64 (m, 1H), 4.36 (s, 2H),4.03 (s, 3H), 4.02 (s, 3H), 3.49-3.39 (m, 2H), 2.35-2.28 (m, 2H),2.00-1.91 (m, 1H), 1.85-1.75 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 168.5,163.2, 158.1, 157.7, 152.1, 148.5, 145.0, 139.6, 134.8, 131.1, 128.6,127.9, 127.9, 126.8, 126.7, 126.6, 105.7, 103.6, 85.2, 55.1, 46.0, 43.0,42.0, 38.6, 30.5, 28.8, 28.6, 23.5. HRMS m/z calculated forC₂₈H₃₀ClN₅O₂(M+H⁺) 504.2161; found 504.2162.

(N1-4′-[1,1′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)5o)

¹H NMR (CD₃ OD; 500 MHz): δ 7.94 (d, J=9.3 Hz, 2H), 7.67-7.63 (m, 4H),7.47-7.43 (m, 2H), 7.12-7.09 (m, 2H), 5.56-5.53 (m, 1H), 5.16 (d, J=45.1Hz, 2H), 3.44-3.35 (m, 2H), 2.29-2.24 (m, 2H), 1.94-1.86 (m, 1H),1.81-1.72 (m, 1H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.7, 163.9, 163.1,163.0, 162.0, 161.7, 161.3, 153.9, 143.8, 136.0, 131.9, 131.4, 128.7,128.6, 128.1, 126.5, 125.7, 115.4, 115.3, 113.8, 109.2, 78.2, 76.9,41.4, 29.1, 23.7. HRMS m/z calculated for C₂₆H₂₅F₂N₅O (M+H⁺) 462.2100;found 462.2097.

(N1-4′-F-[1,1′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(5p)

¹H NMR (CD₃ OD; 500 MHz): δ 7.94 (d, J=9.3 Hz, 2H), 7.68-7.63 (m, 4H),7.61-7.57 (m, 2H), 7.47-7.44 (m, 2H), 7.12-7.08 (m, 2H), 5.57-5.54 (m,1H), 4.28 (s, 2H), 3.42-3.32 (m, 2H), 2.30-2.24 (m, 2H), 1.94-1.86 (m,1H), 1.80-1.72 (m, 1H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.6, 164.0, 163.3,162.0, 162.0, 161.8, 161.4, 153.9, 143.9, 136.0, 135.9, 131.9, 131.3,128.8, 128.7, 128.1, 126.5, 125.8, 117.8, 115.5, 115.3, 113.8, 41.9,38.7, 29.1, 23.6. HRMS m/z calculated for C₂₆H₂₅FClN₅O (M+H⁺) 478.1804;found 478.1803.

(N1-[1,1′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5,6-difluoro-1H-benzo[d]imidazol-2-yl)-L-ornithine)(5q)

¹H NMR (CD₃ OD; 500 MHz): δ 7.92 (d, J=9.4 Hz, 2H), 7.63 (d, J=9.4 Hz,2H), 7.56-7.50 (m, 4H), 7.38-7.34 (m, 2H), 7.29-7.26 (m, 1H), 5.50-5.47(m, 1H), 5.17 (d, J=45.3 Hz, 2H), 3.43-3.34 (m, 2H), 2.27-2.14 (m, 2H),1.91-1.82 (m, 1H), 1.79-1.69 (m, 1H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.6,163.1, 162.9, 161.4, 161.3, 160.8, 156.1, 150.0, 149.9, 148.1, 147.8,144.9, 139.6, 131.6, 129.9, 128.7, 128.0, 127.8, 126.7, 102.3, 102.2,78.3, 76.8, 41.5, 29.5, 23.7. HRMS m/z calculated for C₂₆H₂₄F₃N₅O (M+H⁺)480.2006; found 480.1998.

(N1-[1,1′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5,6-difluoro-1H-benzo[d]imidazol-2-yl)-L-ornithine)(5r)

¹H NMR (CD₃ OD; 500 MHz): δ 7.92 (d, J=9.3 Hz, 2H), 7.63 (d, J=9.4 Hz,2H), 7.56-7.49 (m, 4H), 7.37-7.34 (m, 2H), 7.29-7.26 (m, 1H), 5.50-5.47(m, 1H), 4.27 (s, 2H), 3.38-3.33 (m, 2H), 2.28-2.15 (m, 2H), 1.90-1.82(m, 1H), 1.79-1.71 (m, 1H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.6, 163.3,161.3, 161.0, 156.2, 150.0, 149.8, 148.1, 147.9, 144.9, 139.6, 131.5,129.9, 128.71, 128.0, 127.8, 126.7, 126.7, 102.4, 102.4, 102.3, 102.2,42.0, 38.7, 29.5, 23.6. HRMS m/z calculated for C₂₆H₂₄F₂ClN₅O (M+H⁺)496.1710; found 496.1706.

(N1-4′-F-[1,1′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5,6-difluoro-1H-benzo[d]imidazol-2-yl)-L-omithine)(5s)

¹H NMR (CD₃ OD; 500 MHz): δ 7.93-7.90 (d, J=9.1 Hz, 2H), 7.63-7.60 (d,J=9.1 Hz, 2H), 7.59-7.55 (m, 2H), 7.52-7.49 (m, 2H), 7.11-7.07 (m, 2H),5.49-5.46 (m, 1H), 5.16 (d, J=45.4 Hz, 2H), 3.42-3.33 (m, 2H), 2.28-2.14(m, 2H), 1.92-1.81 (m, 1H), 1.79-1.70 (m, 1H). ¹³C NMR (125 MHz, CD₃ OD)δ 168.6, 163.9, 163.1, 162.8, 162.1, 161.2, 161.0, 156.1, 150.0, 149.8,148.0, 147.8, 143.8, 136.0, 131.6, 130.0, 128.7, 128.6, 128.0, 126.5,115.5, 115.3, 102.4, 102.3, 102.3, 102.2, 78.3, 76.9, 41.4, 29.5, 23.8.HRMS m/z calculated for C₂₆H₂₃F₄N₅O (M+H⁺) 498.1911; found 498.1903.

(N1-4′-F-[1,1′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5,6-difluoro-1H-benzo[d]imidazol-2-yl)-L-omithine)(5t)

¹H NMR (CD₃ OD; 500 MHz): δ 7.91 (d, J=9.4 Hz, 2H), 7.61 (d, J=9.3 Hz,2H), 7.59-7.56 (m, 2H), 7.52-7.50 (m, 2H), 7.11-7.07 (m, 2H), 5.50-5.46(m, 1H), 4.27 (s, 2H), 3.39-3.31 (m, 2H), 2.28-2.14 (m, 2H), 1.92-1.82(m, 1H), 1.79-1.70 (m, 1H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.6, 163.9,163.3, 162.0, 161.2, 160.9, 156.2, 150.0, 149.8, 148.0, 147.8, 143.8,135.9, 131.6, 129.8, 128.7, 128.0, 126.6, 115.4, 115.3, 102.4, 102.3,102.3, 102.2, 42.0, 38.7, 29.6, 23.6. HRMS m/z calculated forC₂₆H₂₃F₃ClN₅O (M+H⁺) 514.1616; found 514.1612.

(N1-4′-ethynyl-[1,1′-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(5u)

¹H NMR (CD₃ OD; 400 MHz): δ 8.08 (d, J=8.4 Hz, 2H), 7.81-7.69 (m, 4H),7.65-7.63 (m, 2H), 7.58-7.57 (m, 4H), 5.67 (t, J=7.6 Hz, 1H), 5.30 (d,J=45.2 Hz, 2H), 3.61 (s, 1H), 3.56-3.48 (m, 2H), 2.41-2.35 (m, 2H),2.07-1.85 (m, 2H). ¹³C NMR (100 MHz, CD₃ OD) δ 170.0, 164.5, 164.4,163.2, 162.8, 155.4, 145.3, 141.2, 133.7, 133.4, 133.2, 129.9, 129.6,128.2, 128.1, 127.2, 123.7, 115.2, 84.0, 79.9, 79.8, 78.1, 71.4, 55.8,43.8, 42.8, 30.6, 25.1. HRMS m/z calculated for C₂₈H₂₆FN₅O (M+H⁺)468.2192; found 468.2191.

(N1-4′-ethynyl-[1,1′-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(5v)

¹H NMR (CD₃ OD; 400 MHz): δ 8.07 (d, J=8.4 Hz, 2H), 7.82-7.78 (m, 4H),7.72-7.69 (m, 2H), 7.60-7.57 (m, 4H), 5.67 (t, J=7.6 Hz, 1H), 4.39 (s,2H), 3.61 (s, 1H), 3.52-3.47 (m, 2H), 2.42-2.36 (m, 2H), 2.07-1.83 (m,2H). ¹³C NMR (100 MHz, CD₃ OD) δ 170.0, 164.8, 155.3, 145.3, 141.2,133.7, 133.3, 133.2, 129.6, 128.2, 128.1, 127.3, 123.7, 115.2, 84.0,79.9, 43.4, 40.1, 30.5, 25.0. HRMS m/z calculated for C₂₈H₂₆ClN₅O (M+H⁺)484.1896; found 484.1902.

(N1-4-ethynyl-benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(5w)

¹H NMR (CD₃ OD; 500 MHz): δ 8.13-8.11 (m, 1H), 7.93 (d, J=8.3 Hz, 2H),7.87-7.85 (m, 1H), 7.65-7.62 (m, 2H), 7.58 (d, J=8.3 Hz, 2H), 5.80-5.77(m, 1H), 5.35-5.29 (m, 1H), 5.30 (d, J=45.5 Hz, 2H), 3.74 (s, 1H),3.58-3.48 (m, 2H), 2.50-2.42 (m, 1H), 2.34-2.26 (m, 1H), 2.11-2.02 (m,1H), 1.94-1.87 (m, 1H), 1.85 (d, J=6.8 Hz, 3H), 1.79 (d, J=7.2 Hz, 3H).¹³C NMR (125 MHz, CD₃ OD) δ 167.9, 163.1, 163.0, 161.1, 152.1, 132.4,131.8, 131.5, 130.2, 127.5, 126.6, 126.3, 125.9, 114.8, 114.6, 81.9,80.3, 78.3, 76.9, 51.4, 46.3, 41.4, 28.9, 23.8, 19.6. HRMS m/zcalculated for C₂₅H₂₈FN₅O (M+H⁺) 434.2349; found 434.2348.

4. General Procedure for Synthesis of Benzimidazole haloacetamidines6a-j.^(a)

To a stirred solution of 4a-d, g (1.0 eq) in THF was added phthalicanhydride (1.0 eq) and allowed to stir at rt under N₂ for 18 h. Solventswere evaporated and the crude product was purified by reverse phase HPLCusing MeCN:H₂O (0.5% TFA) as the eluent to give the product in 78-86%yield. This product was then treated with TFA to remove the Boc groupgiving the desired 2-CO₂H-Bz-Orn-benzimidazole intermediate. The solventwas then evaporated to dryness and the crude material was dried invacuo. To a stirred solution of the corresponding2-CO₂H-Bz-Orn-benzimidazole intermediate in dry MeOH was added TEA (4.0eq) followed by ethyl haloacetimidate HCl (2.0 eq). The reaction wasstirred under N₂ at rt for 3 h. Solvents were then evaporated underreduced pressure and the crude product was purified by reverse phaseHPLC using MeCN:H₂O (0.5% TFA) as an eluent to give compounds 6a-j in52-69% yield.

(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(6a)

¹H NMR (CD₃ OD; 400 MHz): δ 8.06 (dd, J=1.2 Hz, J=8.0 Hz, 2H), 7.84-7.79(m, 2H), 7.71-7.67 (m, 1H), 7.63-7.56 (m, 4H), 5.59-5.54 (m, 1H), 5.26(d, J=45.2 Hz, 2H), 3.52-3.48 (m, 2H), 2.38-2.23 (m, 2H), 2.09-1.89 (m,2H). ¹³C NMR (100 MHz, CD₃ OD) δ 173.5, 169.4, 164.8, 162.9, 162.6,154.6, 139.0, 133.6, 133.3, 131.7, 131.2, 130.3, 128.9, 127.2, 115.2,79.8, 78.1, 42.8, 30.5, 25.1. HRMS m/z calculated for C₂₁H₂₃FN₅O₃(M+H⁺-H₂O) 394.1674; found 394.1673.

(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-omithine)(6b)

¹H NMR (CD₃ OD; 400 MHz): δ 8.07 (dd, J=1.2 Hz, J=8.0 Hz, 2H), 7.83-7.79(m, 2H), 7.72-7.67 (m, 1H), 7.64-7.57 (m, 4H), 5.57-5.54 (m, 1H), 4.41(s, 2H), 3.50-3.46 (m, 2H), 2.39-2.24 (m, 2H), 2.09-1.88 (m, 2H). ¹³CNMR (100 MHz, CD₃ OD) δ 173.5, 169.4, 164.8, 162.9, 162.6, 154.6, 139.0,133.6, 133.3, 131.7, 131.2, 130.3, 128.9, 127.2, 115.2 (2C), 43.3, 40.1,30.2 (2C), 24.9. HRMS m/z calculated for C₂₁H₂₃ClN₅O₃(M+H⁺- H₂O)410.1378; found 410.1382.

(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(6c)

¹H NMR (CD₃ OD; 400 MHz): δ 7.99 (dd, J=1.4 Hz, J=7.8 Hz, 1H), 7.86 (dd,J=2.2 Hz, J=7.1 Hz, 1H), 7.80 (dd, J=2.2 Hz, J=7.2 Hz, 1H), 7.66-7.54(m, 4H), 7.49 (dd, J=1.4 Hz, J=7.5 Hz, 1H), 5.68-5.64 (m, 1H), 5.28 (d,J=45.3 Hz, 2H), 4.19 (s, 3H), 3.52-3.46 (m, 2H), 2.41-2.31 (m, 1H),2.29-2.21 (m, 1H), 2.09-1.99 (m, 1H), 1.96-1.86 (m, 1H). ¹³C NMR (100MHz, CD₃ OD) δ 171.7, 166.0, 160.6, 156.0, 151.9, 137.4, 131.8, 130.0,129.7, 127.3, 125.7, 125.5, 114.9, 111.8, 110.5, 78.3, 76.6, 45.3, 41.3,30.6, 28.3, 23.6. HRMS m/z calculated for C₂₂H₂₄FN₅O₃(M+H⁺) 426.1936;found 426.1938.

(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(6d)

¹H NMR (CD₃ OD; 400 MHz): δ 7.99 (dd, J=1.4 Hz, J=7.5 Hz, 1H), 7.83 (dd,J=2.3 Hz, J=7.2 Hz, 1H), 7.78 (dd, J=1.8 Hz, J=7.2 Hz, 1H), 7.66-7.54(m, 4H), 7.48 (dd, J=1.4 Hz, J=7.4 Hz, 1H), 5.67-5.63 (m, 1H), 4.38 (s,2H), 4.17 (s, 3H), 3.49-3.45 (m, 2H), 2.41-2.23 (m, 2H), 2.07-1.98 (m,1H), 1.94-1.87 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.1, 167.3, 161.9,152.1, 137.5, 133.9, 133.7, 132.0, 130.2, 129.6, 127.3, 125.3, 115.2,111.6, 109.9, 108.9, 101.1, 100.5, 45.3, 42.0, 30.6, 23.5. HRMS m/zcalculated for C₂₂H₂₄ClN₅O₃(M+H⁺) 442.1640; found 442.1642.

(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(6e)

¹H NMR (CD₃ OD; 400 MHz): δ 7.99 (dd, J=1.6 Hz, J=7.4 Hz, 1H), 7.90 (dd,J=2.2 Hz, J=7.4 Hz, 1H), 7.82 (dd, J=2.2 Hz, J=7.3 Hz, 1H), 7.66-7.54(m, 4H), 7.48 (dd, J=1.6 Hz, J=7.4 Hz, 1H), 5.66-5.62 (m, 1H), 5.28 (d,J=45.5 Hz, 2H), 4.70-4.61 (m, 2H), 3.52-3.41 (m, 2H), 2.40-2.20 (m, 2H),2.09-1.98 (m, 1H), 1.94-1.83 (m, 1H), 1.59 (t, J=7.1 Hz, 3H). ¹³C NMR(100 MHz, CD₃ OD) δ 171.3, 167.1, 160.1, 151.5, 137.2, 131.8, 129.8,129.6, 128.8, 128.4, 126.8, 1260.0, 125.6, 125.6, 114.9, 111.9, 78.0,45.1, 41.2, 40.2, 29.0, 23.4, 13.2. HRMS m/z calculated forC₂₃H₂₆FN₅O₃(M+H⁺) 440.2092; found 440.2092.

(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(6f)

¹H NMR (CD₃ OD; 400 MHz): δ 7.99 (dd, J=1.3 Hz, J=7.4 Hz, 1H), 7.88 (dd,J=1.7 Hz, J=7.1 Hz, 1H), 7.81 (dd, J=2.1 Hz, J=7.1 Hz, 1H), 7.65-7.54(m, 4H), 7.47 (dd, J=1.7 Hz, J=7.1 Hz, 1H), 5.65-5.61 (m, 1H), 4.68-4.59(m, 2H), 4.37 (s, 2H), 3.47-3.41 (m, 2H), 2.40-2.21 (m, 2H), 2.09-1.98(m, 1H), 1.95-1.85 (m, 1H), 1.60 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz,CD₃ OD) δ 171.2, 167.0, 151.5, 137.3, 137.1, 132.1, 129.7, 129.1, 128.7,127.8, 127.2, 125.3, 125.0, 115.1, 111.5, 87.0, 45.0, 41.7, 38.5, 28.5,28.1, 23.3, 13.5. HRMS m/z calculated for C₂₃H₂₆ClN₅O₃(M+H⁺) 456.1797;found 456.1799.

(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(62)

¹H NMR (CD₃ OD; 400 MHz): δ 8.05-8.02 (m, 1H), 7.99 (dd, J=1.7 Hz, J=7.6Hz, 1H), 7.82-7.78 (m, 2H), 7.65-7.52 (m, 4H), 7.47 (dd, J=1.7 Hz, J=7.5Hz, 1H), 5.67-5.63 (m, 1H), 5.27 (d, J=45.3 Hz, 2H), 5.31-5.25 (m, 1H),3.53-3.43 (m, 2H), 2.37-2.20 (m, 2H), 2.07-1.96 (m, 1H), 1.93-1.85 (m,1H), 1.82 (d, J=2.4 Hz, 3H), 1.80 (d, J=2.3 Hz, 3H). ¹³C NMR (100 MHz,CD₃ OD) δ 171.4, 167.5, 151.3, 137.1, 131.8, 130.1, 129.6, 129.1, 127.2,125.9, 124.9, 115.7, 115.6, 114.1, 110.0, 78.3, 76.7, 50.5, 45.6, 41.4,29.0, 23.5, 19.9, 19.8. HRMS m/z calculated for C₂₄H₂₈FN₅O₃(M+H⁺)454.2249; found 454.2249.

(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(6h)

¹H NMR (CD₃ OD; 400 MHz): δ 8.10-8.07 (m, 1H), 7.99 (dd, J=1.7 Hz, J=7.4Hz, 1H), 7.86-7.81 (m, 1H), 7.66-7.54 (m, 4H), 7.48 (dd, J=1.6 Hz, J=7.4Hz, 1H), 5.70-5.66 (m, 1H), 5.37-5.30 (m, 1H), 4.37 (s, 2H), 3.47-3.43(m, 2H), 2.39-2.19 (m, 2H), 2.09-1.98 (m, 1H), 1.93-1.82 (m, 1H), 1.84(d, J=2.5 Hz, 3H), 1.82 (d, J=2.5 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ171.6, 167.4, 163.2, 151.1, 136.9, 131.8, 130.4, 130.1, 129.5, 129.1,127.2, 125.7, 125.3, 114.9, 114.5, 111.0, 50.8, 45.7, 41.7, 38.4, 28.9,23.5, 19.8, 19.8. HRMS m/z calculated for C₂₄H₂₈ClN₅O₃(M+H⁺) 470.1953;found 470.1959.

(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(6i)

¹H NMR (CD₃ OD; 400 MHz): δ 7.98 (dd, J=1.6 Hz, J=7.6 Hz, 1H), 7.65-7.61(m, 1H), 7.57-7.47 (m, 3H), 7.40 (d, J=8.2 Hz, 1H), 7.11 (d, J=8.2 Hz,1H), 5.63-5.59 (m, 1H), 5.27 (d, J=45.4 Hz, 2H), 4.17 (s, 3H), 4.05 (s,3H), 3.50-3.45 (m, 2H), 2.40-2.30 (m, 1H), 2.29-2.18 (m, 1H), 2.08-1.96(m, 1H), 1.89-1.77 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.6, 166.7,162.8, 160.9, 150.8, 148.2, 137.4, 134.5, 131.7, 129.8, 129.3, 128.6,127.1, 126.6, 105.6, 103.4, 78.0, 76.2, 55.0, 45.5, 41.3, 30.5, 28.1,23.2. HRMS m/z calculated for C₂₃H₂₆FN₅O₄(M+H⁺) 456.2042; found456.2039.

(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(6j)

¹H NMR (CD₃ OD; 400 MHz): δ 7.99 (dd, J=1.6 Hz, J=7.6 Hz, 1H), 7.65-7.61(m, 1H), 7.58-7.47 (m, 3H), 7.39 (d, J=8.3 Hz, 1H), 7.10 (d, J=8.1 Hz,1H), 5.63-5.59 (m, 1H), 4.39 (s, 2H), 4.16 (s, 3H), 4.05 (s, 3H),3.47-3.43 (m, 2H), 2.40-2.30 (m, 1H), 2.29-2.20 (m, 1H), 2.08-1.96 (m,1H), 1.89-1.78 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.7, 167.2, 163.2,150.9, 147.9, 137.2, 134.5, 132.0, 130.0, 129.6, 128.8, 127.2, 126.9,115.0, 106.0, 103.6, 55.1, 45.6, 42.0, 38.4, 31.1, 28.0, 23.5. HRMS m/zcalculated for C₂₃H₂₆FN₅O₄(M+H⁺) 472.1745; found 472.1746.

3. General Procedure for Synthesis of Benzimidazole haloacetamidines7a-v.^(a)

To a stirred solution of 4a-d, f-j (1.0 eq) in DMF was added HOBt (2.0eq), HBTU (2.0 eq), and DIPEA (3.0 eq) followed by3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (1.0 eq) and allowed tostir at rt for 12 h. The reaction mixture was then diluted with water.The product was filtered, washed with water, dried under vacuum, andobtained in 62-71% yield. This product was then treated with 2 M HCl inEt₂O to remove the Boc group giving the Oxoisoindoline-Orn-benzimidazoleintermediate. The solvent was then evaporated to dryness and the crudematerial was dried in vacuo. To a stirred solution of the correspondingOxoisoindoline-Om-benzimidazole intermediate in dry MeOH was added TEA(4.0 eq) followed by ethyl haloacetimidate HCl (2.0 eq). The reactionwas stirred under N₂ at rt for 3 h. Solvents were then evaporated underreduced pressure and the crude product was purified by reverse phaseHPLC using MeCN:H₂O (0.5% TFA) as an eluent to give compounds 7a-v in41-57% yield.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(7a)

¹H NMR (CD₃ OD; 400 MHz): δ 8.18 (d, J=8.3 Hz, 1H), 7.83 (dd, J=1.6 Hz,J=8.3 Hz, 1H), 7.79-7.74 (m, 3H), 7.59-7.55 (m, 2H), 5.59-5.56 (m, 1H),5.29 (d, J=45.3 Hz, 2H), 4.59 (s, 2H), 3.53-3.45 (m, 2H), 2.38-2.27 (m,2H), 2.04-1.91 (m, 2H). ¹³C NMR (100 MHz, CD₃ OD) δ 170.1, 166.6, 163.1,154.3, 146.2, 131.8, 130.7, 130.2, 130.0, 128.8, 127.1, 126.9, 125.9,118.0, 113.6, 102.5, 78.4, 76.7, 45.5, 41.2, 29.6, 23.7. HRMS m/zcalculated for C₂₂H₂₃FN₆O₂(M+H⁺) 423.1939; found 423.1941.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(7b)

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (d, J=8.2 Hz, 1H), 7.83 (dd, J=1.6 Hz,J=7.7 Hz, 1H) 7.79-7.70 (m, 3H), 7.54-7.51 (m, 2H), 5.58-5.54 (m, 1H),4.59 (s, 2H), 4.39 (s, 2H), 3.48-3.43 (m, 2H), 2.44-2.28 (m, 2H),2.04-1.91 (m, 2H). ¹³C NMR (100 MHz, CD₃ OD) δ 172.0, 163.3, 154.1,146.1, 131.8, 131.5, 130.7, 130.2, 130.0, 128.8, 127.9, 127.1, 125.9,125.8, 113.5, 110.0, 45.5, 43.1, 41.8, 38.8, 29.5, 23.6. HRMS m/zcalculated for C₂₂H₂₃ClN₆O₂(M+H⁺) 439.1644; found 439.1646.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(7c)

¹H NMR (CD₃ OD; 400 MHz): δ 8.18 (dd, J=1.3 Hz, J=7.5 Hz, 1H), 7.91 (dd,J=1.6 Hz, J=7.2 Hz, 1H), 7.82 (dd, J=1.6 Hz, J=7.5 Hz, 1H), 7.75 (t,J=7.8 Hz, 2H), 7.67-7.58 (m, 2H), 5.65-5.62 (m, 1H), 5.30 (d, J=45.4 Hz,2H), 4.58 (s, 2H), 4.21 (s, 3H), 3.54-3.44 (m, 2H), 2.37-2.25 (m, 2H),2.13-2.03 (m, 1H), 2.02-1.93 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.9,166.2, 153.2, 146.3, 132.9, 131.7, 130.3, 130.1, 128.8, 127.2, 126.4,126.0, 113.8, 112.3, 109.8, 78.5, 76.7, 45.4, 41.2, 37.3, 30.7, 28.6,23.6. HRMS m/z calculated for C₂₃H₂₅FN₆O₂(M+H⁺) 437.2096; found437.2095.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(7d)

¹H NMR (CD₃ OD; 400 MHz): δ 8.18 (dd, J=1.3 Hz, J=7.6 Hz, 1H), 7.91 (dd,J=1.2 Hz, J=7.5 Hz, 1H), 7.82 (dd, J=1.4 Hz, J=7.6 Hz, 1H), 7.74 (t,J=7.8 Hz, 2H), 7.66-7.58 (m, 2H), 5.66-5.62 (m, 1H), 4.58 (s, 2H), 4.41(s, 2H), 4.21 (s, 3H), 3.51-3.44 (m, 2H), 2.37-2.26 (m, 2H), 2.13-1.93(m, 2H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.9, 166.3, 163.4, 153.4, 146.3,132.9, 131.8, 130.8, 130.3, 130.1, 128.9, 127.2, 126.4, 126.0, 114.0,112.2, 46.7, 45.4, 41.8, 38.8, 30.7, 28.7, 23.6. HRMS m/z calculated forC₂₃H₂₅ClN₆O₂(M+H⁺) 453.1800; found 453.1799.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7e)

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (dd, J=1.6 Hz, J=7.6 Hz, 1H), 7.94 (dd,J=1.6 Hz, J=7.4 Hz, 1H), 7.83 (dd, J=1.2 Hz, J=7.6 Hz, 1H), 7.77-7.72(m, 2H), 7.66-7.58 (m, 2H), 5.65-5.62 (m, 1H), 5.30 (d, J=45.4 Hz, 2H),4.78-4.65 (m, 2H), 4.58 (s, 2H), 3.54-3.44 (m, 2H), 2.38-2.22 (m, 2H),2.14-1.95 (m, 2H), 1.62 (t, J=7.4 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ171.9, 168.7, 165.9, 164.2, 156.4, 152.5, 145.9, 143.4, 131.6, 130.3,130.1, 128.7, 127.7, 127.1, 126.3, 125.9, 114.1, 112.3, 45.3, 41.2,40.4, 29.2, 23.8, 13.2. HRMS m/z calculated for C₂₄H₂₇FN₆O₂(M+H⁺)451.2252; found 451.2250.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7f)

¹H NMR (CD₃ OD; 400 MHz): δ 8.19 (dd, J=1.5 Hz, J=7.3 Hz, 1H), 7.94 (dd,J=1.5 Hz, J=7.5 Hz, 1H), 7.82 (dd, J=1.3 Hz, J=7.3 Hz, 1H), 7.77-7.72(m, 2H), 7.66-7.58 (m, 2H), 5.66-5.62 (m, 1H), 4.78-4.65 (m, 2H), 4.58(s, 2H), 4.41 (s, 2H), 3.52-3.43 (m, 2H), 2.38-2.23 (m, 2H), 2.15-1.96(m, 2H), 1.62 (t, J=7.4 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.8,166.1, 163.3, 159.2, 158.2, 153.0, 146.3, 131.8, 130.3, 128.8, 127.4,126.3, 126.0, 116.5, 114.1, 112.4, 72.1, 45.4, 41.9, 40.5, 38.8, 29.2,23.6, 13.3. HRMS m/z calculated for C₂₄H₂₇ClN₆O₂(M+H⁺) 467.1957; found467.1952.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-omithine)(7g)

¹H NMR (CD₃OD; 500 MHz): δ 8.21 (dd, J=1.5 Hz, J=7.7 Hz, 1H), 8.14 (dd,J=1.5 Hz, J=7.5 Hz, 1H), 7.85 (dd, J=1.4 Hz, J=7.4 Hz, 1H), 7.80-7.76(m, 2H), 7.66-7.61 (m, 2H), 5.73-5.70 (m, 1H), 5.37-5.31 (m, 1H), 5.34(d, J=45.3 Hz, 2H), 4.61 (s, 2H), 3.60-3.49 (m, 2H), 2.40-2.22 (m, 2H),2.15-2.07 (m, 1H), 2.06-1.99 (m, 1H), 1.88 (d, J=1.8 Hz, 3H), 1.86 (d,J=1.6 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.8, 166.3, 163.2, 160.5,160.1, 152.8, 146.4, 131.9, 131.3, 130.4, 130.3, 128.9, 127.2, 126.2,125.7, 114.8, 114.3, 78.3, 77.0, 51.3, 45.5, 41.1, 29.3, 23.8, 19.5.HRMS m/z calculated for C₂₅H₂₉FN₆O₂(M+H⁺) 465.2409; found 465.2409.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(7h)

¹H NMR (CD₃ OD; 500 MHz): δ 8.21 (dd, J=1.3 Hz, J=7.4, Hz, 1H), 8.14(dd, J=2.0 Hz, J=7.6 Hz, 1H), 7.86 (dd, J=1.6 Hz, J=7.3 Hz, 2H),7.80-7.76 (m, 2H), 7.66-7.61 (m, 2H), 5.73-5.70 (m, 1H), 5.37-5.32 (m,1H), 4.61 (s, 2H), 4.45 (s, 2H), 3.57-3.47 (m, 2H), 2.4-2.25 (m, 2H),2.16-2.08 (m, 1H), 2.07-1.98 (m, 1H), 1.88 (d, J=3.6 Hz, 3H), 1.86 (d,J=3.5 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.9, 166.3, 163.4, 160.6,160.4, 152.7, 146.2, 131.9, 131.3, 130.3, 130.2, 128.9, 127.3, 126.2,125.8, 114.7, 114.4, 51.3, 47.2, 45.4, 41.8, 38.7, 29.3, 23.7, 19.5.HRMS m/z calculated for C₂₅H₂₉ClN₆O₂(M+H⁺) 481.2113; found 481.2112.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1H-benzo[d]imidazol-2-yl)-L-omithine)(7i)

¹H NMR (CD₃ OD; 400 MHz): δ 8.10 (dd, J=9.2 Hz, 1H), 7.73 (dd, J=9.3 Hz,1H), 7.68-7.65 (m, 1H), 7.38-7.35 (m, 1H), 7.17 (dd, J=9.4 Hz, 1H), 6.98(dd, J=9.2 Hz, 1H), 5.41-5.38 (m, 1H), 5.18 (d, J=45.5 Hz, 2H), 4.49 (s,2H), 3.95 (s, 3H), 3.41-3.34 (m, 2H), 2.23-2.18 (m, 2H), 1.93-1.79 (m,2H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.9, 166.5, 161.2, 153.6, 147.8,146.1, 133.0, 131.8, 130.6, 130.2, 128.8, 127.1, 126.9, 105.9, 105.3,100.0, 78.3, 76.9, 55.3, 45.5, 41.3, 29.9, 23.7. HRMS m/z calculated forC₂₃H₂₅FN₆O₃(M+H⁺) 453.2045; found 453.2042.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(7j)

¹H NMR (CD₃ OD; 500 MHz) δ 8.11 (dd, J=1.1 Hz, J=7.8 Hz, 1H), 7.73 (dd,J=1.1 Hz, J=7.6 Hz, 1H), 7.67 (t, J=7.8 Hz 1H), 7.36 (t, J=8.3 Hz, 1H),7.17 (d, J=8.6 Hz, 1H), 6.97 (d, J=8.1 Hz, 1H), 5.41-5.38 (m, 1H), 4.49(s, 2H), 4.29 (s, 2H), 3.95 (s, 3H), 3.39-3.33 (m, 2H), 2.23-2.19 (m,2H), 1.93-1.80 (m, 2H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.4, 166.1, 163.6,154.1, 153.6, 146.1, 131.6, 130.7, 130.3, 129.6, 128.6, 127.2, 126.6,105.8, 105.5, 100.0, 74.3, 55.1, 45.1, 41.8, 38.4, 29.2, 23.5. HRMS m/zcalculated for C₂₃H₂₅ClN₆O₃(M+H⁺) 469.1749; found 469.1750.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(7k)

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (dd, J=1.2 Hz, J=7.8 Hz, 1H), 7.81 (dd,J=1.3 Hz, J=7.6 Hz, 1H), 7.74 (t, J=7.6 Hz, 1H), 7.53 (t, J=8.2 Hz, 1H),7.41 (d, J=8.5 Hz, 1H), 7.10 (d, J=8.2 Hz), 5.60-5.56 (m, 1H), 5.30 (d,J=45.3 Hz, 2H), 4.56 (s, 2H), 4.16 (s, 3H), 4.00 (s, 3H), 3.54-3.43 (m,2H), 2.40-2.23 (m, 2H), 2.12-2.01 (m, 1H), 1.98-1.87 (m, 1H). ¹³C NMR(100 MHz, CD₃ OD) δ 171.8, 166.0, 160.8, 152.4, 148.1, 146.1, 134.3,131.6, 130.3, 130.0, 128.9, 127.2, 126.8, 109.8, 106.2, 103.9, 78.3,76.7, 55.3, 45.4, 41.2, 30.8, 28.8, 23.6. HRMS m/z calculated forC₂₄H₂₇FN₆O₃ (M+H⁺) 467.2201; found 467.2201.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(7l)

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (dd, J=1.3 Hz, J=7.5 Hz, 1H), 7.77 (dd,J=1.3 Hz, J=7.5 Hz, 1H), 7.74 (t, J=7.6 Hz, 1H), 7.53 (t, J=8.2 Hz, 1H),7.40 (d, J=8.4 Hz, 1H), 7.09 (d, J=8.1 Hz, 1H), 5.61-5.57 (m, 1H), 4.56(s, 2H), 4.40 (s, 2H), 4.15 (s, 3H), 4.00 (s, 3H), 3.48-3.44 (m, 2H),2.40-2.23 (m, 2H), 2.11-2.01 (m, 1H), 1.98-1.87 (m, 1H). ¹³C NMR (100MHz, CD₃ OD) δ 171.8, 165.9, 163.4, 160.8, 152.4, 148.4, 146.2, 134.4,132.8, 131.8, 130.4, 130.2, 128.9, 127.3, 126.9, 106.2, 103.7, 55.3,45.4, 41.8, 38.7, 30.7, 28.7, 23.6. HRMS m/z calculated forC₂₄H₂₇ClN₆O₃(M+H⁺) 483.1906; found 483.1907.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-ethoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7m)

¹H NMR (CD₃ OD; 50O MHz): δ 8.10 (dd, J=1.1 Hz, J=7.7 Hz, 1H), 7.71 (dd,J=1.1 Hz, J=7.7 Hz, 1H), 7.64 (t, J=7.7 Hz, 1H), 7.41 (t, J=8.2 Hz, 1H),7.30 (d, J=8.1 Hz, 1H), 6.97 (d, J=8.4 Hz, 1H), 5.50-5.47 (m, 1H), 5.21(d, J=45.4 Hz, 2H), 4.46 (s, 2H), 4.18-4.13 (m, 2H), 4.08 (s, 3H),3.44-3.36 (m, 2H), 2.32-2.23 (m, 1H), 2.22-2.14 (m, 1H), 2.02-1.94 (m,1H), 1.89-1.79 (m, 1H), 1.36 (t, J=7.1 Hz, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 171.9, 166.0, 163.0, 161.3, 152.6, 147.2, 146.3, 134.3, 131.8,130.5, 130.2, 128.9, 127.3, 127.1, 107.1, 103.7, 78.4, 76.9, 64.5, 45.4,41.3, 31.0, 28.8, 23.8, 13.5. HRMS m/z calculated for C₂₅H₂₉FN₆O₃(M+H⁺)481.2358; found 481.2356.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-ethoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7n)

¹H NMR (CD₃ OD; 500 MHz): δ 8.08 (dd, J=1.2 Hz, J=7.7 Hz, 1H), 7.70 (dd,J=1.1 Hz, J=7.6 Hz, 1H), 7.63 (t, J=7.7 Hz, 1H), 7.42 (t, J=8.3 Hz, 1H),7.29 (d, J=8.1 Hz, 1H), 6.97 (d, J=8.1 Hz, 1H), 5.51-5.47 (m, 1H), 4.45(s, 2H), 4.32 (s, 2H), 4.17-4.12 (m, 2H), 4.08 (s, 3H), 3.40-3.36 (m,2H), 2.33-2.24 (m, 1H), 2.23-2.16 (m, 1H), 2.03-1.94 (m, 1H), 1.89-1.80(m, 1H), 1.34 (t, J=7.1 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.9,166.1, 163.5, 161.2, 160.8, 152.6, 147.1, 146.3, 134.3, 131.8, 130.4,130.2, 129.0, 127.3, 121.5, 107.2, 103.7, 64.6, 45.4, 41.9, 38.8, 30.9,28.8, 23.7, 13.4. HRMS m/z calculated for C₂₅H₂₉ClN₆O₃(M+H⁺) 497.2062;found 497.2061.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(7o)

¹H NMR (CD₃ OD; 500 MHz): δ 8.08 (dd, J=1.2 Hz, J=7.6 Hz, 1H), 7.68 (m,3H), 7.15 (dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.08 (d, J=2.4 Hz, 1H),5.50-5.47 (m, 1H), 5.21 (d, J=45.2 Hz, 2H), 4.48 (s, 2H), 4.08 (s, 3H),3.78 (s, 3H), 3.45-3.35 (m, 2H), 2.27-2.13 (m, 2H), 2.02-1.93 (m, 1H),1.91-1.82 (m, 1H). ¹³C NMR (125 MHz, CD₃ OD) δ 172.0, 166.3, 161.1,159.5, 152.2, 146.3, 131.8, 131.5, 130.2, 128.9, 127.3, 127.0, 116.2,113.0, 95.9, 78.3, 76.9, 55.2, 46.6, 45.5, 41.2, 30.7, 28.8, 23.8. HRMSm/z calculated for C₂₄H₂₇FN₆O₃(M+H⁺) 467.2201; found 467.2201.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(⁷p)

¹H NMR (CD₃ OD; 500 MHz): δ 8.08 (dd, J=1.1 Hz, J=7.5 Hz, 1H), 7.68 (m,3H), 7.16 (dd, J=2.5 Hz, J=9.2 Hz, 1H), 7.08 (d, J=2.3 Hz, 1H),5.51-5.48 (m, 1H), 4.48 (s, 2H), 4.08 (s, 3H), 3.78 (s, 3H), 3.43-3.33(m, 2H), 2.28-2.14 (m, 2H), 2.01-1.93 (m, 1H), 1.91-1.82 (m, 1H). ¹³CNMR (125 MHz, CD₃ OD) δ 171.9, 166.3, 163.5, 160.8, 159.4, 152.2, 146.3,131.9, 131.5, 130.2, 128.9, 127.3, 127.0, 116.2, 113.0, 95.9, 55.2,46.6, 45.5, 41.9, 38.7, 30.8, 28.7, 23.6. HRMS m/z calculated forC₂₄H₂₇ClN₆O₃(M+H⁺) 483.1906; found 483.1906.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7q)

¹H NMR (CD₃OD; 500 MHz): δ 8.12 (dd, J=1.1 Hz, J=7.7 Hz, 1H), 7.72 (dd,J=1.1 Hz, J=7.6 Hz, 1H), 7.65 (t, J=7.2 Hz, 1H), 7.44 (t, J=8.4 Hz, 1H),7.34 (d, J=8.4 Hz, 1H), 7.00 (d, J=8.1 Hz, 1H), 5.52-5.48 (m, 1H), 5.21(d, J=45.3 Hz, 2H), 4.64-4.57 (m, 1H), 4.56-4.50 (m, 1H), 4.48 (s, 2H),3.92 (s, 3H), 3.45-3.35 (m, 2H), 2.29-2.21 (m, 1H), 2.20-2.13 (m, 1H),2.03-1.94 (m, 1H), 1.90-1.83 (m, 1H), 1.49 (t, J=7.2 Hz, 3H). ¹³C NMR(125 MHz, CD₃ OD) δ 172.0, 165.9, 161.1, 152.1, 148.2, 146.3, 133.3,131.9, 130.5, 130.2, 128.9, 127.3, 127.0, 106.4, 104.0, 78.4, 77.0,55.3, 46.8, 45.5, 41.4, 40.6, 29.4, 23.8, 13.4. HRMS m/z calculated forC₂₅H₂₉FN₆O₃(M+H⁺) 481.2358; found 481.2353.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7r)

¹H NMR (CD₃ OD; 500 MHz): δ 8.11 (dd, J=1.3 Hz, J=7.9 Hz, 1H), 7.72 (dd,J=1.3 Hz, J=7.6 Hz, 1H), 7.64 (t, J=7.6 Hz, 1H), 7.45 (t, J=8.3 Hz, 1H),7.35 (d, J=8.4 Hz, 1H), 7.02 (d, J=8.2 Hz, 1H), 5.52-5.49 (m, 1H),4.64-4.58 (m, 1H), 4.57-4.52 (m, 1H), 4.48 (s, 2H), 4.31 (s, 2H), 3.91(s, 3H), 3.42-3.34 (m, 2H), 2.30-2.22 (m, 1H), 2.20-2.12 (m, 1H),2.04-1.95 (m, 1H), 1.91-1.82 (m, 1H), 1.49 (t, J=7.5 Hz, 3H). ¹³C NMR(125 MHz, CD₃ OD) δ 172.0, 166.0, 163.4, 160.7, 152.1, 148.1, 146.3,133.1, 131.9, 130.5, 130.2, 128.9, 127.3, 121.9, 106.5, 104.0, 55.3,46.8, 45.4, 41.8, 40.6, 38.8, 29.2, 23.7, 13.3. HRMS m/z calculated forC₂₅H₂₉ClN₆O₃(M+H⁺) 497.2062; found 497.2060.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(7s)

¹H NMR (CD₃ OD; 500 MHz): δ 8.22 (d, J=8.3 Hz, 1H), 7.86 (d, J=8.3 Hz,1H), 7.80 (t, J=6.6 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H), 7.23 (s, 1H), 7.20(dd, J=2.3 Hz, J=8.8 Hz, 1H), 5.58-5.55 (m, 1H), 5.32 (d, J=45.7 Hz,2H), 4.62 (s, 2H), 3.92 (s, 3H), 3.57-3.47 (m, 2H), 2.38-2.28 (m, 2H),2.07-1.93 (m, 2H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.9, 169.3, 166.7,159.0, 153.2, 146.2, 131.9, 130.8, 130.0, 128.9, 127.0, 115.9, 114.4,99.8, 95.7, 78.3, 76.7, 55.2, 45.4, 41.2, 29.6, 23.7. HRMS m/zcalculated for C₂₃H₂₅FN₆O₃(M+H⁺) 453.2045; found 453.2037.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(7t)

¹H NMR (CD₃ OD; 500 MHz): δ 8.05 (d, J=8.1 Hz, 1H), 7.71 (d, J=8.2 Hz,1H), 7.65 (t, J=7.6 Hz, 1H), 7.52 (d, J=8.9 Hz, 1H), 7.11 (s, 1H), 7.07(dd, J=2.2 Hz, J=8.4 Hz, 1H), 5.48-5.45 (m, 1H), 4.48 (s, 2H), 4.31 (s,2H), 3.78 (s, 3H), 3.40-3.33 (m, 2H), 2.26-2.16 (m, 2H), 1.94-1.82 (m,2H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.8, 166.8, 163.5, 159.2, 153.2,146.1, 132.2, 131.9, 130.8, 130.0, 128.8, 127.0, 125.2, 116.2, 114.4,95.8, 55.0, 45.4, 41.8, 38.8, 29.6, 23.5. HRMS m/z calculated forC₂₃H₂₅ClN₆O₃(M+H⁺) 469.1749; found 469.1741.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7u)

¹H NMR (CD₃ OD; 500 MHz): δ 8.20 (d, J=7.7 Hz, 1H), 7.84 (d, J=8.4 Hz,2H), 7.77 (t, J=7.7 Hz, 1H), 7.26 (dd, J=2.2 Hz, J=8.4 Hz, 1H), 7.22 (d,J=2.3 Hz, 1H), 5.64-5.61 (m, 1H), 5.34 (d, J=45.5 Hz, 2H), 4.78-4.64 (m,2H), 4.61 (s, 2H), 3.90 (s, 3H), 3.59-3.48 (m, 2H), 2.41-2.24 (m, 2H),2.19-1.98 (m, 2H), 1.84-1.80 (m, 1H), 1.75-1.69 (m, 1H), 1.64 (t, J=7.2Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.9, 166.2, 163.2, 161.1, 159.4,151.8, 146.3, 131.8, 130.4, 128.9, 127.2, 125.7, 116.2, 113.3, 96.1,78.3, 77.0, 55.0, 46.7, 45.5, 41.4, 40.7, 29.4, 23.7, 13.4. HRMS m/zcalculated for C₂₅H₂₉FN₆O₃(M+H⁺) 481.2358; found 481.2358.

(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7v)

¹H NMR (CD₃ OD; 500 MHz): δ 8.07 (d, J=8.0 Hz, 1H), 7.71 (d, J=8.6 Hz,2H), 7.64 (t, J=7.9 Hz, 1H), 7.13 (dd, J=2.6 Hz, J=8.8 Hz, 1H), 7.08 (d,J=2.3 Hz, 1H), 5.51-5.48 (m, 1H), 4.64-4.50 (m, 2H), 4.47 (s, 2H), 4.33(s, 2H), 3.77 (s, 3H), 3.42-3.35 (m, 2H), 2.28-2.11 (m, 2H), 2.03-1.83(m, 2H), 1.72-1.67 (m, 1H), 1.62-1.58 (m, 1H), 1.51 (t, J=8.1 Hz, 3H).¹³C NMR (125 MHz, CD₃ OD) δ 171.9, 166.2, 163.4, 161.2, 161.0, 159.5,151.9, 146.3, 131.8, 130.3, 128.8, 127.2, 125.8, 125.6, 116.2, 113.2,96.0, 55.2, 46.6, 45.5, 41.8, 40.6, 38.8, 29.4, 23.7, 13.4. HRMS m/zcalculated for C₂₅H₂₉ClN₆O₃(M+H⁺) 497.2062; found 497.2065.

5. General Procedure for Synthesis of Benzimidazole haloacetamidines8a-b′.^(a)

To a stirred solution of 4a-d, f-j (1.0 eq) in DMF was added HOBt (2.0eq), HBTU (2.0 eq), and DIPEA (3.0 eq) followed by2-alkyl-3-oxo-4-isoindoline carboxylic acid (1.0 eq) and allowed to stirat rt for 12 h. The reaction mixture was then diluted with water. Theproduct was filtered, washed with water, dried under vacuum, andobtained in 62-71% yield. This product was then treated with 2 M HCl inEt₂O to remove the Boc group giving theN-alkyl-Oxoisoindoline-Orn-benzimidazole intermediate. The solvent wasthen evaporated to dryness and the crude material was dried in vacuo. Toa stirred solution of the correspondingN-alkyl-Oxoisoindoline-Om-benzimidazole intermediate in dry MeOH wasadded TEA (4.0 eq) followed by ethyl haloacetimidate HCl (2.0 eq). Thereaction was stirred under N₂ at rt for 3 h. Solvents were thenevaporated under reduced pressure and the crude product was purified byreverse phase HPLC using MeCN:H₂O (0.5% TFA) as an eluent to givecompounds 8a-b′ in 56-74% yield.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(8a)

¹H NMR (CD₃ OD; 400 MHz): δ 8.11 (dd, J=1.1 Hz, J=7.7 Hz, 1H), 7.80-7.70(m, 4H), 7.60-7.55 (m, 2H), 5.64-5.59 (m, 1H), 5.28 (d, J=45.4 Hz, 2H),4.63 (s, 2H), 3.80-3.74 (m, 2H), 3.52-3.48 (m, 2H), 2.39-2.28 (m, 2H),2.06-1.92 (m, 2H), 1.35 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ168.3, 166.4, 163.1, 162.9, 160.7, 154.0, 143.3, 131.3, 131.1, 130.2,129.8, 129.1, 126.4, 126.1 (2C), 113.5, 78.3, 76.6, 49.2, 41.2, 37.5,29.5, 23.7, 11.9. HRMS m/z calculated for C₂₄H₂₇FN₆O₂(M+H⁺) 451.2252;found 451.2249.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(8b)

¹H NMR (CD₃OD; 400 MHz): δ 8.12 (dd, J=1.2 Hz, J=7.8 Hz, 1H), 7.81-7.70(m, 4H), 7.60-7.56 (m, 2H), 5.63-5.59 (m, 1H), 4.63 (s, 2H), 4.38 (s,2H), 3.80-3.75 (m, 2H), 3.50-3.45 (m, 2H), 2.41-2.31 (m, 2H), 2.08-1.92(m, 2H), 1.35 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ 168.3,166.5, 163.2, 161.1, 160.8, 154.1, 143.4, 131.3, 131.1, 130.2, 129.8,129.0, 126.5, 125.9 (2C), 113.5 (2C), 49.2, 41.7, 38.7, 37.4, 29.4,23.4, 12.0. HRMS m/z calculated for C₂₄H₂₇ClN₆O₂(M+H⁺) 467.1957; found467.1954.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8c)

¹H NMR (CD₃ OD; 400 MHz): δ 8.17 (dd, J=1.2 Hz, J=7.9 Hz, 1H), 7.88 (dd,J=1.5 Hz, J=7.7 Hz, 1H), 7.80 (dd, J=1.2 Hz, J=7.8 Hz, 1H), 7.74-7.70(m, 2H), 7.64-7.56 (m, 2H), 5.67-5.63 (m, 1H), 5.28 (d, J=45.5 Hz, 2H),4.64 (s, 2H), 4.19 (s, 3H), 3.79-3.72 (m, 2H), 3.56-3.45 (m, 2H),2.41-2.25 (m, 2H), 2.14-2.03 (m, 1H), 2.01-1.89 (m, 1H), 1.34 (t, J=7.5Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ 168.1, 166.0, 162.9, 158.3, 152.8,143.4, 133.1, 131.3, 130.3, 129.8, 129.3, 126.8, 126.1, 125.6, 114.1,111.9, 109.8, 78.4, 76.5, 49.2, 41.3, 37.5, 30.5, 28.8, 23.6, 12.0. HRMSm/z calculated for C₂₅H₂₉FN₆O₂(M+H⁺) 465.2409; found 465.2404.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8d)

¹H NMR (CD₃ OD; 400 MHz): δ 8.16 (dd, J=1.5 Hz, J=7.7 Hz, 1H), 7.91 (dd,J=1.1 Hz, J=7.6 Hz, 1H), 7.80 (dd, J=1.5 Hz, J=7.6 Hz, 1H), 7.75-7.70(m, 2H), 7.66-7.58 (m, 2H), 5.68-5.63 (m, 1H), 4.64 (s, 2H), 4.38 (s,2H), 4.21 (s, 3H), 3.79-3.73 (m, 2H), 3.50-3.46 (m, 2H), 2.42-2.26 (m,2H), 2.16-2.05 (m, 1H), 2.02-1.91 (m, 1H), 1.34 (t, J=7.2 Hz, 3H). ¹³CNMR (100 MHz, CD₃ OD) δ 168.4, 165.9, 163.2, 160.8, 153.3, 143.5, 132.5,131.2, 130.1, 129.5, 129.2, 126.7, 126.3, 125.7, 113.9, 112.1, 109.9,49.2, 41.7, 38.7, 37.5, 30.6, 28.7, 23.4, 11.9. HRMS m/z calculated forC₂₅H₂₉ClN₆O₂(M+H⁺) 481.2113; found 481.2118.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(8e)

¹H NMR (CD₃ OD; 400 MHz): δ 8.15 (dd, J=1.1 Hz, J=7.8 Hz, 1H), 7.95 (dd,J=1.2 Hz, J=7.2 Hz, 1H), 7.80 (dd, J=1.2 Hz, J=7.7 Hz, 1H), 7.76-7.69(m, 2H), 7.67-7.59 (m, 2H), 5.67-5.63 (m, 1H), 5.29 (d, J=45.4 Hz, 2H),4.82-4.66 (m, 2H), 4.64 (s, 2H), 3.81-3.72 (m, 3H), 3.56-3.46 (m, 2H),2.42-2.23 (m, 2H), 2.18-2.08 (m, 1H), 2.05-1.93 (m, 1H), 1.64 (t, J=7.2Hz, 3H), 1.35 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ 168.1,165.9, 163.0, 153.1, 143.5, 131.6, 131.3, 130.6, 130.1, 129.6, 129.2,126.7, 126.4, 126.1, 113.9, 112.5, 78.4, 76.7, 49.2, 41.2, 40.6, 37.6,29.2, 23.8, 13.2, 12.1. HRMS m/z calculated for C₂₆H₃₁FN₆O₂(M+H⁺)479.2565; found 479.2565.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(8f)

¹H NMR (CD₃ OD; 400 MHz): δ 8.15 (dd, J=1.2 Hz, J=7.8 Hz, 1H), 7.94 (dd,J=1.1 Hz, J=7.1 Hz, 1H), 7.80 (dd, J=1.1 Hz, 7.4 Hz, 1H), 7.76-7.69 (m,2H), 7.66-7.58 (m, 2H), 5.68-5.64 (m, 1H), 4.81-4.66 (m, 2H), 4.63 (s,2H), 4.40 (s, 2H), 3.79-3.74 (m, 3H), 3.52-3.47 (m, 2H), 2.43-2.33 (m,1H), 2.31-2.25 (m, 1H), 2.18-2.07 (m, 1H), 2.05-1.94 (m, 1H), 1.63 (t,J=7.3 Hz, 3H), 1.34 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ168.3, 166.3, 163.1, 161.2, 152.8, 143.8, 131.6, 131.2, 130.5, 130.0,129.6, 129.0, 126.7, 126.3, 125.7, 114.0, 112.2, 49.2, 42.0, 40.4, 38.5,37.4, 29.0, 23.3, 13.3, 11.9. HRMS m/z calculated for C₂₆H₃₁ClN₆O₂(M+H⁺)495.2270; found 495.2273.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(8g)

¹H NMR (CD₃ OD; 400 MHz): δ 8.16-8.10 (m, 2H), 7.81-7.69 (m, 3H),7.62-7.57 (m, 2H), 5.72-5.68 (m, 1H), 5.34-5.29 (m, 1H), 5.28 (d, J=45.4Hz, 2H), 4.64 (s, 2H), 3.80-3.73 (m, 2H), 3.56-3.47 (m, 2H), 2.42-2.21(m, 2H), 2.16-2.05 (m, 1H), 2.03-1.92 (m, 1H), 1.85 (d, J=2.7 Hz, 3H),1.83 (d, J=2.7 Hz, 3H), 1.35 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 168.4, 166.1, 163.2, 162.7, 161.2, 160.8, 152.7, 143.6, 131.4,130.1, 129.7, 129.1, 126.6, 126.0, 125.6, 114.7, 114.3, 78.4, 76.6,51.2, 49.2, 41.3, 37.4, 29.3, 23.8, 19.4, 12.0. HRMS m/z calculated forC₂₇H₃₃FN₆O₂(M+H⁺) 493.2722; found 493.2723.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(8h)

¹H NMR (CD₃ OD; 400 MHz): δ 8.16-8.09 (m, 2H), 7.81-7.69 (m, 3H),7.61-7.57 (m, 2H), 5.72-5.68 (m, 1H), 5.36-5.29 (m, 1H), 4.64 (s, 2H),4.40 (s, 2H), 3.79-3.74 (m, 2H), 3.52-3.48 (m, 2H), 2.42-2.22 (m, 2H),2.17-2.06 (m, 1H), 2.05-1.92 (m, 1H), 1.85 (d, J=3.4 Hz, 3H), 1.83 (d,J=3.4 Hz, 3H), 1.34 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ168.5, 166.3, 163.4, 161.2, 160.9, 160.5, 152.8, 143.6, 131.3, 130.2,129.8, 129.2, 126.8, 126.2, 125.7, 115.0, 114.7, 114.3, 51.3, 49.3,41.9, 38.7, 37.5, 29.4, 23.6, 19.6, 12.1. HRMS m/z calculated forC₂₇H₃₃ClN₆O₂(M+H⁺) 509.2426; found 509.2426.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(8i)

¹H NMR (CD₃ OD; 500 MHz): δ 8.04 (d, J=7.8 Hz, 1H), 7.70 (d, J=7.9 Hz,1H), 7.63 (t, J=8.7 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.19 (d, J=8.7 Hz,1H), 6.99 (d, J=8.7 Hz, 1H), 5.45-5.42 (m, 1H), 5.18 (d, J=45.3 Hz, 2H),4.55 (s, 2H), 3.95 (s, 3H), 3.71-3.64 (m, 2H), 3.43-3.37 (m, 2H),2.26-2.21 (m, 2H), 1.96-1.79 (m, 2H), 1.26 (t, J=7.9 Hz, 3H). ¹³C NMR(125 MHz, CD₃ OD) δ 168.6, 166.7, 163.2, 153.5, 147.7, 143.6, 132.6,131.4, 130.4, 130.0, 129.2, 127.2, 126.6, 122.0, 106.2, 105.4, 78.3,76.8, 55.3, 49.4, 41.4, 37.5, 29.7, 23.8, 12.1. HRMS m/z calculated forC₂₅H₂₉FN₆O₃(M+H) 481.2358; found 481.2358.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(8j)

¹H NMR (CD₃ OD; 500 MHz): δ 8.16 (d, J=7.4 Hz, 1H), 7.83 (d, J=7.2 Hz,1H), 7.76 (d, J=7.8 Hz, 1H), 7.52 (t, J=7.5 Hz, 1H), 7.32 (d, J=8.1 Hz,1H), 7.12 (d, J=7.8 Hz, 1H), 5.58-5.55 (m, 1H), 4.67 (s, 2H), 4.41 (s,2H), 4.07 (s, 3H), 3.83-3.79 (m, 2H), 3.52-3.48 (m, 2H), 2.39-2.34 (m,2H), 2.09-1.93 (m, 2H), 1.38 (t, J=7.5 Hz, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 168.6, 166.5, 163.5, 153.7, 147.6, 143.5, 132.6, 131.3, 130.4,129.9, 129.1, 127.3, 126.7, 125.6, 121.9, 106.2, 105.2, 55.3, 49.4,41.9, 38.8, 37.6, 29.7, 23.6, 11.9. HRMS m/z calculated forC₂₅H₂₉ClN₆O₃(M+H⁺) 497.2062; found 497.2060.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8k)

¹H NMR (CD₃ OD; 400 MHz): δ 8.16 (dd, J=1.4 Hz, J=7.8 Hz, 1H), 7.80 (dd,J=1.3 Hz, J=7.8 Hz, 1H), 7.71 (t, J=7.5 Hz, 1H), 7.55 (t, J=8.3 Hz, 1H),7.42 (dd, J=1.1 Hz, J=8.6 Hz, 1H), 7.11 (d, J=8.2 Hz, 1H), 5.62-5.58 (m,1H), 5.19 (d, J=45.5 Hz, 2H), 4.63 (s, 2H), 4.18 (s, 3H), 4.01 (s, 3H),3.81-3.70 (m, 2H), 3.52-3.48 (m, 2H), 2.43-2.24 (m, 2H), 2.14-2.03 (m,1H), 1.98-1.86 (m, 1H), 1.34 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 168.5, 166.1, 160.7, 152.5, 147.9, 143.7, 134.4, 131.3, 130.3,129.7, 129.3, 127.1, 126.7, 115.0, 106.5, 103.9, 78.4, 76.6, 55.3, 49.2,41.3, 37.6, 30.9, 28.8, 23.8, 12.0. HRMS m/z calculated forC₂₆H₃₁FN₆O₃(M+H⁺) 495.2514; found 495.2512.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8l)

¹H NMR (CD₃ OD; 400 MHz): δ 8.17 (dd, J=1.1 Hz, J=7.8 Hz, 1H), 7.79 (dd,J=1.3 Hz, J=7.9 Hz, 1H), 7.72 (t, J=7.6 Hz, 1H), 7.56 (t, J=8.4 Hz, 1H),7.43 (dd, J=1.1 Hz, J=8.7 Hz, 1H), 7.12 (d, J=8.1 Hz, 1H), 5.62-5.59 (m,1H), 4.63 (s, 2H), 4.39 (s, 2H), 4.19 (s, 3H), 4.01 (s, 3H), 3.82-3.71(m, 2H), 3.50-3.46 (m, 2H), 2.44-2.26 (m, 2H), 2.15-2.05 (m, 1H),2.00-1.87 (m, 1H), 1.34 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ168.4, 166.1, 163.3, 152.5, 148.4, 147.8, 143.7, 134.2, 131.4, 130.2,129.9, 129.2, 127.2, 126.8, 106.5, 103.8, 55.2, 49.4, 46.8, 41.9, 38.6,37.5, 30.9, 28.7, 23.6, 11.9. HRMS m/z calculated for C₂₆H₃₁ClN₆O₃(M+H⁺)511.2219; found 511.2220.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-ethoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8m)

¹H NMR (CD₃ OD; 500 MHz): δ 8.06 (dd, J=1.1 Hz, J=8.1 Hz, 1H), 7.69 (dd,J=1.2 Hz, J=7.6 Hz, 1H), 7.60 (t, J=7.5 Hz, 1H), 7.42 (t, J=8.4 Hz, 1H),7.30 (dd, J=1.0 Hz, J=8.2 Hz, 1H), 6.98 (d, J=8.2 Hz, 1H), 5.52-5.48 (m,1H), 5.18 (d, J=45.6 Hz, 2H), 4.52 (s, 2H), 4.19-4.14 (m, 2H), 4.08 (s,3H), 3.70-3.62 (m, 2H), 3.43-3.39 (m, 2H), 2.35-2.26 (m, 1H), 2.24-2.16(m, 1H), 2.04-1.95 (m, 1H), 1.87-1.78 (m, 1H), 1.36 (t, J=7.1 Hz, 3H),1.25 (t, J=7.1 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.5, 166.2, 152.5,147.2, 143.8, 134.3, 131.4, 130.3, 129.8, 129.4, 127.1, 126.7, 121.6,107.2, 103.7, 78.3, 76.9, 64.5, 49.3, 46.8, 41.5, 37.5, 30.9, 28.8,23.8, 13.4, 12.0. HRMS m/z calculated for C₂₇H₃₃FN₆O₃(M+H⁺) 509.2671;found 509.2676.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2chloro-1-iminoethyl)-1-(4-ethoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8n)

¹H NMR (CD₃ OD; 500 MHz): δ 8.05 (dd, J=1.1 Hz, J=8.0 Hz, 1H), 7.68 (dd,J=1.1 Hz, J=7.6 Hz, 1H), 7.60 (t, J=7.8 Hz, 1H), 7.41 (t, J=8.2 Hz, 1H),7.28 (dd, J=1.0 Hz, J=8.6 Hz, 1H), 6.96 (d, J=8.2 Hz, 1H), 5.52-5.49 (m,1H), 4.51 (s, 2H), 4.30 (s, 2H), 4.17-4.12 (m, 2H), 4.08 (s, 3H),3.68-3.64 (m, 2H), 3.40-3.37 (m, 2H), 2.35-2.26 (m, 1H), 2.25-2.18 (m,1H), 2.05-1.95 (m, 1H), 1.87-1.78 (m, 1H), 1.35 (t, J=7.2 Hz, 3H), 1.25(t, J=7.3 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.5, 166.2, 163.3,152.6, 147.3, 143.6, 134.4, 131.4, 130.3, 129.8, 129.3, 127.0, 126.6,121.9, 107.2, 103.7, 64.6, 49.2, 46.9, 42.0, 38.7, 37.5, 30.9, 28.9,23.7, 13.5, 12.1. HRMS m/z calculated for C₂₇H₃₃ClN₆O₃(M+H⁺) 525.2375;found 525.2378.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8o)

¹H NMR (CD₃ OD; 500 MHz): δ 8.06 (dd, J=1.2 Hz, J=7.9 Hz, 1H), 7.70 (m,2H), 7.63-7.59 (t, J=7.9 Hz, 1H), 7.14 (dd, J=2.4 Hz, J=9.2 Hz, 1H),7.09 (d, J=2.3 Hz, 1H), 5.52-5.49 (m, 1H), 5.18 (d, J=45.3 Hz, 2H), 4.53(s, 2H), 4.09 (s, 3H), 3.77 (s, 3H), 3.69-3.64 (m, 2H), 3.47-3.37 (m,2H), 2.31-2.16 (m, 2H), 2.04-1.95 (m, 1H), 1.90-1.81 (m, 1H), 1.25 (t,J=7.4 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.6, 166.2, 161.0, 159.6,152.2, 143.7, 131.4, 130.3, 129.8, 129.3, 127.0, 126.7, 116.3, 113.1,95.9, 78.3, 76.9, 55.1, 49.3, 46.7, 41.4, 37.5, 30.9, 28.7, 23.8, 12.0.HRMS m/z calculated for C₂₆H₃₁FN₆O₃(M+H⁺) 495.2514; found 495.2514.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8p)

¹H NMR (CD₃ OD; 500 MHz): δ 8.05 (dd, J=1.1 Hz, J=7.7 Hz, 1H), 7.70-7.67(m, 2H), 7.61 (t, J=7.6 Hz, 1H), 7.13 (dd, J=2.5 Hz, J=9.1 Hz, 1H), 7.09(d, J=2.4 Hz, 1H), 5.52-5.49 (m, 2H), 4.52 (s, 2H), 4.30 (s, 3H), 4.09(s, 3H), 3.77 (s, 3H), 3.69-3.63 (m, 2H), 3.41-3.38 (m, 2H), 2.32-2.16(m, 2H), 2.05-1.96 (m, 1H), 1.91-1.82 (m, 1H), 1.24 (t, J=7.2 Hz, 3H).¹³C NMR (125 MHz, CD₃ OD) δ 168.6, 166.3, 163.4, 160.8, 152.2, 143.7,131.4, 130.2, 129.8, 129.3, 127.0, 126.7, 125.7, 116.2, 113.1, 95.9,55.2, 49.3, 46.8, 41.9, 38.7, 37.5, 30.9, 28.8, 23.6, 12.0. HRMS m/zcalculated for C₂₆H₃₁ClN₆O₃(M+H⁺) 511.2219; found 511.2218.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(8q)

¹H NMR (CD₃ OD; 500 MHz): δ 8.06 (d, J=7.9 Hz, 1H), 7.70 (d, J=8.0 Hz,1H), 7.61 (t, J=8.2 Hz, 1H), 7.44 (t, J=8.2 Hz, 1H), 7.31 (d, J=8.2 Hz,1H), 7.01 (d, J=8.0 Hz, 1H), 5.52-5.49 (m, 1H), 5.18 (d, J=45.2 Hz, 2H),4.57-4.51 (m, 1H), 4.49 (s, 2H), 4.07 (s, 3H), 3.91 (s, 3H), 3.44-3.37(m, 2H), 2.33-2.25 (m, 1H), 2.24-2.17 (m, 1H), 2.04-1.95 (m, 1H),1.87-1.78 (m, 1H), 1.29 (d, J=2.8, 3H), 1.28 (d, J=2.7 Hz, 3H). ¹³C NMR(125 MHz, CD₃ OD) δ 168.2, 166.1, 152.6, 148.0, 143.8, 134.5, 131.3,130.3, 129.9, 129.5, 127.1, 126.8, 121.9, 106.5, 103.9, 78.3, 76.9,55.3, 46.8, 45.4, 44.1, 41.4, 30.9, 28.8, 23.8, 19.3, 19.2. HRMS m/zcalculated for C₂₇H₃₃FN₆O₃(M+H⁺) 509.2669; found 509.2674.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(8r)

¹H NMR (CD₃ OD; 500 MHz): δ 8.03 (d, J=8.1 Hz, 1H), 7.68 (d, J=7.7 Hz,1H), 7.58 (t, J=7.8 Hz, 1H), 7.43 (t, J=7.9 Hz, 1H), 7.31 (d, J=8.1 Hz,1H), 6.99 (d, J=8.1 Hz, 1H), 5.53-5.50 (m, 1H), 4.59-4.51 (m, 1H), 4.47(s, 2H), 4.31 (s, 2H), 4.09 (s, 3H), 3.89 (s, 3H), 3.41-3.38 (m, 2H),2.34-2.27 (m, 1H), 2.25-2.18 (m, 1H), 2.05-1.96 (m, 1H), 1.89-1.79 (m,1H), 1.29 (d, J=2.5 Hz, 3H), 1.27 (d, J=2.5 Hz, 3H). ¹³C NMR (125 MHz,CD₃ OD) δ 168.1, 166.2, 163.4, 152.6, 147.8, 143.8, 134.2, 131.3, 130.2,129.8, 129.5, 127.3, 126.8, 121.2, 106.7, 104.0, 55.4, 46.9, 43.6, 44.0,42.0, 38.7, 31.1, 28.8, 23.7, 19.3, 19.3. HRMS m/z calculated forC₂₇H₃₃ClN₆O₃(M+H⁺) 525.2374; found 525.2381.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-cyclopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(8s)

¹H NMR (CD₃ OD; 500 MHz): δ 8.03 (d, J=8.2 Hz, 1H), 7.64 (d, J=8.1 Hz,1H), 7.58 (t, J=7.5 Hz, 1H), 7.44 (t, J=8.2 Hz, 1H), 7.31 (d, J=8.1 Hz,1H), 6.99 (d, J=8.2 Hz, 1H), 5.52-5.49 (m, 1H), 5.20 (d, J=45.5 Hz, 2H),4.42 (s, 2H), 4.09 (s, 3H), 3.90 (s, 3H), 3.47-3.37 (m, 2H), 3.02-2.98(m, 1H), 2.34-2.26 (m, 1H), 2.25-2.17 (m, 1H), 2.04-1.95 (m, 1H),1.88-1.79 (m, 1H), 0.93-0.84 (m, 4H). ¹³C NMR (125 MHz, CD₃ OD) δ 170.3,166.1, 152.6, 147.9, 143.7, 134.3, 131.5, 130.3, 129.8, 129.4, 127.3,126.7, 121.4, 106.6, 104.0, 78.4, 76.9, 55.4, 50.0, 46.9, 41.4, 31.1,28.8, 25.6, 23.8, 4.7, 4.5. HRMS m/z calculated for C₂₇H₃₁FN₆O₃(M+H⁺)507.2512; found 507.2516.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-cyclopropyl-benzo[d]imidazol-2-yl)-L-omithine)(8t)

¹H NMR (CD₃ OD; 500 MHz): δ 8.02 (d, J=8.0 Hz, 1H), 7.63 (d, J=8.1 Hz,1H), 7.58 (t, J=8.1 Hz, 1H), 7.43 (t, J=8.2 Hz, 1H), 7.31 (d, J=8.2 Hz,1H), 6.99 (d, J=8.2 Hz, 1H), 5.53-5.49 (m, 1H), 4.42 (s, 2H), 4.32 (s,2H), 4.09 (s, 3H), 3.90 (s, 3H), 3.42-3.38 (m, 2H), 3.02-2.98 (m, 1H),2.35-2.27 (m, 1H), 2.25-2.18 (m, 1H), 2.05-1.96 (m, 1H), 1.88-1.79 (m,1H), 0.92-0.85 (m, 4H). ¹³C NMR (125 MHz, CD₃ OD) δ 170.3, 166.2, 163.5,152.6, 147.8, 143.7, 134.2, 131.5, 130.2, 129.7, 129.3, 127.3, 126.6,121.3, 106.6, 103.9, 55.3, 49.9, 46.9, 41.8, 38.8, 31.0, 28.7, 25.6,23.6, 4.8, 4.5. HRMS m/z calculated for C₂₇H₃₁ClN₆O₃(M+H⁺) 523.2216;found 523.2225.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(8u)

¹H NMR (CD₃ OD; 500 MHz): δ 8.07 (dd, J=1.5 Hz, J=7.9 Hz, 1H), 7.69 (dd,J=1.5 Hz, J=7.4 Hz, 1H), 7.61 (t, J=7.7 Hz, 1H), 7.44 (t, J=8.1 Hz, 1H),7.34 (dd, J=1.0 Hz, J=8.2 Hz, 1H), 7.01 (d, J=8.3 Hz, 1H), 5.53-5.49 (m,1H), 5.19 (d, J=45.2 Hz, 2H), 4.66-4.58 (m, 1H), 4.57-4.54 (m, 1H), 4.53(s, 2H), 3.91 (s, 3H), 3.73-3.61 (m, 2H), 3.43-3.39 (m, 2H), 2.33-2.25(m, 1H), 2.22-2.14 (m, 1H), 2.05-1.96 (m, 1H), 1.90-1.80 (m, 1H), 1.49(t, J=7.2 Hz, 3H), 1.25 (t, J=7.4 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ168.6, 166.0, 152.2, 148.1, 143.7, 133.3, 131.4, 130.3, 129.8, 129.2,127.2, 126.8, 121.8, 106.6, 104.1, 78.2, 76.9, 55.2, 49.2, 46.9, 41.5,40.7, 37.7, 29.3, 23.9, 13.3, 12.2. HRMS m/z calculated forC₂₇H₃₃FN₆O₃(M+H⁺) 509.2671; found 509.2671.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(8v)

¹H NMR (CD₃ OD; 500 MHz): δ 8.06 (dd, J=1.4 Hz, J=8.1 Hz, 1H), 7.69 (dd,J=1.3 Hz, J=8.1 Hz, 1H), 7.60 (t, J=7.7 Hz, 1H), 7.45 (t, J=8.4 Hz, 1H),7.34 (dd, J=1.0 Hz, J=8.2 Hz, 1H), 7.00 (d, J=7.7 Hz, 1H), 5.53-5.50 (m,1H), 4.65-4.59 (m, 1H), 4.58-4.53 (m, 1H), 4.52 (s, 2H), 4.30 (s, 2H),3.90 (s, 3H), 3.71-3.62 (m, 2H), 3.41-3.37 (m, 2H), 2.33-2.25 (m, 1H),2.23-2.15 (m, 1H), 2.06-1.97 (m, 1H), 1.91-1.81 (m, 1H), 1.50 (t, J=7.4Hz, 3H), 1.25 (t, J=7.2 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.5,166.1, 163.3, 152.2, 148.0, 143.8, 133.2, 131.4, 130.4, 129.8, 129.3,127.3, 126.8, 121.9, 106.5, 104.0, 55.4, 49.3, 46.9, 42.0, 40.7, 38.8,37.7, 29.4, 23.8, 13.3, 12.1. HRMS m/z calculated for C₂₇H₃₃ClN₆O₃(M+H⁺)525.2375; found 525.2374.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(7w)

¹H NMR (500 MHz, CD₃ OD) δ 8.03 (dd, J=2.5 Hz, J=8.7 Hz, 1H), 7.69 (d,J=8.1 Hz, 1H), 7.62 (t, J=8.2 Hz, 1H), 7.53 (d, J=8.8 Hz, 1H), 7.12 (s,1H), 7.07 (dd, J=2.3 Hz, J=9.1 Hz, 1H), 5.49-5.46 (m, 1H), 5.18 (d,J=45.1 Hz, 2H), 4.53 (s, 2H), 3.78 (s, 3H), 3.69-3.65 (m, 2H), 3.42-3.38(m, 1H), 3.27-3.22 (m, 1H), 1.95-1.79 (m, 2H), 1.24 (t, J=8.4 Hz, 3H).¹³C NMR (125 MHz, CD₃ OD) δ 168.6, 166.7, 161.7, 161.4, 161.2, 159.0,153.4, 143.6, 132.3, 131.4, 130.3, 129.9, 129.1, 126.5, 125.3, 116.0,114.3, 95.7, 78.2, 76.7, 55.0, 49.2, 44.2, 41.3, 38.8, 37.5, 29.6, 26.0,23.7, 12.0. HRMS m/z calculated for C₂₅H₂₉FN₆O₃(M+H⁺) 481.2358; found481.2357.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(7x)

¹H NMR (500 MHz, CD₃ OD) δ 8.02 (t, J=6.5 Hz, 1H), 7.68 (d, J=8.1 Hz,1H), 7.61 (t, J=7.6 Hz, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.11 (s, 1H), 7.06(dd, J=2.1 Hz, J=8.6 Hz, 1H), 5.50-5.47 (m, 1H), 4.52 (s, 3H), 4.29 (s,2H), 3.77 (s, 3H), 3.69-3.63 (m, 2H), 3.40-3.36 (m, 1H), 3.28-3.22 (m,1H), 2.27-2.19 (m, 2H), 1.95-1.80 (m, 2H), 1.24 (t, J=6.9 Hz, 3H). ¹³CNMR (125 MHz, CD₃ OD) δ 168.4, 166.7, 163.4, 161.7, 161.2, 161.0, 159.1,153.5, 143.6, 133.2, 131.3, 130.3, 129.9, 129.1, 126.4, 125.7, 125.2,116.1, 114.3, 95.8, 55.1, 49.3, 44.1, 41.9, 38.8, 37.5, 29.7, 29.6,25.9, 23.5, 22.4, 12.1. HRMS m/z calculated for C₂₅H₂₉ClN₆O₃(M+H⁺)497.2062; found 497.2063.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7y)

¹H NMR (500 MHz, CD₃ OD) δ 8.19 (d, J=8.2 Hz, 1H), 7.82 (t, J=6.6 Hz,2H), 7.73 (t, J=7.8 Hz, 1H), 7.26-7.22 (m, 2H), 5.65-5.62 (m, 1H), 5.32(d, J=45.6 Hz, 2H), 4.78-4.67 (m, 2H), 4.65 (s, 2H), 3.90 (s, 3H),3.82-3.77 (m, 2H), 3.60-3.51 (m, 2H), 2.46-2.37 (m, 1H), 2.35-2.27 (m,1H), 2.19-2.10 (m, 1H), 2.05-1.96 (m, 1H), 1.64 (t, J=8.0 Hz, 3H), 1.37(t, J=7.4 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.5, 166.2, 163.1,163.0, 159.3, 152.0, 143.7, 131.9, 131.3, 130.3, 129.8, 129.3, 126.8,125.8, 116.3, 113.2, 96.1, 78.3, 76.9, 55.1, 49.3, 46.7, 41.5, 40.6,37.5, 29.3, 23.8, 13.4, 12.1. HRMS m/z calculated for C₂₇H₃₃FN₆O₃(M+H⁺)509.2671; found 509.2670.

(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(7z)

¹H NMR (500 MHz, CD₃ OD) δ 8.06 (d, J=7.7 Hz, 1H), 7.70 (t, J=7.0 Hz,2H), 7.61 (t, J=7.7 Hz, 1H), 7.14-7.09 (m, 2H), 5.53-5.49 (m, 1H),4.65-4.54 (m, 2H), 4.53 (s, 2H), 4.30 (s, 2H), 3.77 (s, 3H), 3.69-3.64(m, 2H), 3.42-3.38 (m, 2H), 2.32-2.24 (m, 1H), 2.22-2.15 (m, 1H),2.06-1.98 (m, 1H), 1.93-1.84 (m, 1H), 1.51 (t, J=7.0 Hz, 3H), 1.24 (t,J=7.0 Hz, 3H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.5, 166.2, 163.4, 161.0,159.5, 151.9, 143.7, 131.9, 131.4, 130.3, 129.8, 129.3, 126.8, 125.9,116.3, 113.3, 96.1, 55.1, 49.3, 46.7, 41.9, 40.7, 38.7, 37.5, 29.3,23.6, 13.4, 12.0. HRMS m/z calculated for C₂₇H₃₃ClN₆O₃(M+H⁺) 525.2375;found 525.2378.

(N1-(2-methyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(8a′)

¹H NMR (500 MHz, CD₃ OD) δ 8.05 (d, J=8.1 Hz, 1H), 7.67 (d, J=8.1 Hz,1H), 7.59 (t, J=7.4 Hz, 1H), 7.45 (t, J=9.1 Hz, 1H), 7.32 (d, J=9.1 Hz,1H), 7.01 (d, J=8.1 Hz, 1H), 5.52-5.49 (m, 1H), 5.19 (d, J=45.6 Hz, 2H),4.50 (s, 2H), 4.09 (s, 3H), 3.90 (s, 3H), 3.44-3.39 (m, 2H), 3.18 (s,3H), 2.34-2.26 (m, 1H), 2.23-2.16 (m, 1H), 2.05-1.96 (m, 1H), 1.88-1.80(m, 1H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.9, 166.1, 163.2, 163.0, 161.1,160.8, 152.5, 147.8, 143.6, 134.2, 131.4, 130.3, 129.6, 129.1, 127.3,126.6, 121.3, 106.7, 104.0, 78.3, 76.9, 55.3, 51.8, 46.8, 41.4, 31.0,29.0, 28.7, 23.8. HRMS m/z calculated for C₂₅H₂₉FN₆O₃(M+H⁺) 481.2358;found 481.2359.

(N1-(2-methyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(8b′)

¹H NMR (500 MHz, CD₃ OD) δ 8.05 (d, J=7.6 Hz, 1H), 7.67 (d, J=7.7 Hz,1H), 7.59 (t, J=7.4 Hz, 1H), 7.44 (t, J=8.3 Hz, 1H), 7.32 (d, J=8.3 Hz,1H), 7.01 (d, J=8.8 Hz, 1H), 5.52-5.49 (m, 1H), 4.50 (s, 2H), 4.31 (s,2H), 4.09 (s, 3H), 3.90 (s, 3H), 3.41-3.38 (m, 2H), 3.18 (s, 3H),2.34-2.26 (m, 1H), 2.25-2.17 (m, 1H), 2.05-1.96 (m, 1H), 1.89-1.80 (m,1H). ¹³C NMR (125 MHz, CD₃ OD) δ 168.9, 166.1, 163.4, 161.1, 160.7,152.6, 147.8, 143.6, 134.3, 131.4, 130.3, 129.7, 129.2, 127.3, 126.6,121.3, 106.7, 103.9, 55.3, 51.8, 46.8, 41.9, 38.7, 31.1, 29.0, 28.8,23.7. HRMS m/z calculated for C₂₅H₂₉ClN₆O₃(M+H⁺) 497.2062; found497.2062.

6. General Procedure for Synthesis of Benzimidazole haloacetamidines9af.^(a)

To a stirred solution of 4b-d (1.0 eq) in DMF was added HOBt (2.0 eq),HBTU (2.0 eq), and DIPEA (3.0 eq) followed by2-(tert-butoxycarbonyl)benzoic acid (1.0 eq) and allowed to stir at rtfor 12 h. The reaction mixture was then diluted with water. The productwas filtered, washed with water, dried under vacuum, and obtained in62-71% yield. This product was then treated with 1 M HCl in EtOAc toremove the Boc group giving the CO₂ ^(t)Bu-Bz-Orn-benzimidazoleintermediate. The solvent was then evaporated to dryness and the crudematerial was dried in vacuo. To a stirred solution of the correspondingCO₂ ^(t)Bu-Bz-Orn-benzimidazole intermediate in dry MeOH was added TEA(4.0 eq) followed by ethyl haloacetimidate HCl (2.0 eq). The reactionwas stirred under N₂ at rt for 3 h. Solvents were then evaporated underreduced pressure and the crude product was purified by reverse phaseHPLC using MeCN:H₂O (0.5% TFA) as an eluent to give compounds 9a-f in51-67% yield.

(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine(9a)

¹H NMR (CD₃ OD; 400 MHz): δ 7.85-7.81 (m, 2H), 7.77 (dd, J=1.7 Hz, J=7.2Hz, 1H), 7.63-7.51 (m, 4H), 7.48 (dd, J=1.3 Hz, J=7.6 Hz, 1H), 5.68-5.65(m, 1H), 5.27 (d, J=45.3 Hz, 2H), 4.19 (s, 3H), 3.54-3.46 (m, 2H),2.42-2.23 (m, 2H), 2.09-1.98 (m, 1H), 1.95-1.85 (m, 1H), 1.32 (s, 9H).¹³C NMR (100 MHz, CD₃ OD) δ 170.9, 165.0, 151.4, 150.0, 144.4, 143.8,131.5, 130.4, 129.8, 129.5, 125.4, 125.2, 111.7, 100.2, 82.7, 81.0,78.4, 76.1, 54.0, 53.2, 45.1, 40.6, 30.6, 28.4, 26.4, 23.2. HRMS m/zcalculated for C₂₆H₃₂FN₅O₃(M+H⁺) 482.2562; found 482.2561.

(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(9b)

¹H NMR (CD₃ OD; 400 MHz): δ 7.89-7.78 (m, 3H), 7.63-7.46 (m, 5H),5.69-5.64 (m, 1H), 4.37 (s, 2H), 4.21 (s, 3H), 3.50-3.44 (m, 2H),2.43-2.23 (m, 2H), 2.11-1.99 (m, 1H), 1.96-1.85 (m, 1H), 1.32 (s, 9H).¹³C NMR (100 MHz, CD₃ OD) δ 165.3, 163.6, 152.3, 136.4, 131.5, 131.3,130.4, 129.7, 129.3, 126.9, 125.3, 114.7, 111.5, 94.6, 81.2, 72.6, 72.0,66.5, 50.0, 44.8, 42.0, 38.5, 30.2, 28.4, 26.3, 23.3. HRMS m/zcalculated for C₂₆H₃₂ClN₅O₃(M+H⁺) 498.2266; found 498.2268.

(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(9c)

¹H NMR (CD₃ OD; 400 MHz): δ 7.97 (dd, J=2.3 Hz, J=6.8 Hz, 1H), 7.85-7.81(m, 2H), 7.68-7.58 (m, 3H), 7.56-7.49 (m, 2H), 5.70-5.65 (m, 1H), 5.26(d, J=45.3 Hz, 2H), 4.89-4.81 (m, 1H), 4.76-4.68 (m, 1H), 3.50-3.45 (m,2H), 2.42-2.20 (m, 2H), 2.12-2.00 (m, 1H), 1.94-1.81 (m, 1H), 1.64 (t,J=7.5 Hz, 3H), 1.32 (s, 9H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.6, 165.2,163.3, 163.2, 162.4, 161.0, 151.7, 136.7, 131.8, 130.4, 129.7, 129.5,127.1, 126.6, 126.3, 114.3, 112.7, 81.6, 78.6, 76.7, 45.6, 41.8, 40.4,28.8, 26.9, 23.8, 13.4. HRMS m/z calculated for C₂₇H₃₄FN₅O₃(M+H⁺)496.2718; found 496.2718.

(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(9d)

¹H NMR (CD₃ OD; 400 MHz): δ 7.98 (dd, J=2.1 Hz, J=7.2 Hz, 1H), 7.86-7.81(m, 2H), 7.68-7.62 (m, 2H), 7.60 (dd, J=1.3 Hz, J=7.5 Hz, 1H), 7.56-7.49(m, 2H), 5.70-5.65 (m, 1H), 4.90-4.82 (m, 1H), 4.77-4.68 (m, 1H), 4.38(s, 2H), 3.48-3.43 (m, 2H), 2.43-2.21 (m, 2H), 2.15-2.02 (m, 1H),1.95-1.83 (m, 1H), 1.64 (t, J=7.3 Hz, 3H), 1.32 (s, 9H). ¹³C NMR (100MHz, CD₃ OD) δ 171.6, 165.3, 163.3, 160.4, 160.1, 159.8, 151.4, 136.1,132.2, 130.5, 130.2, 128.4, 127.3, 126.3, 108.9, 88.7, 81.4, 50.4, 45.9,44.5, 38.2, 36.4, 31.7, 28.4, 27.0, 25.3, 13.8. HRMS m/z calculated forC₂₇H₃₄ClN₅O₃(M+H⁺) 512.2423; found 512.2422.

(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-omithine)(9e)

¹H NMR (CD₃ OD; 400 MHz): δ 8.12 (m, 1H), 7.86-7.81 (m, 2H), 7.64-7.58(m, 3H), 7.55 (dd, J=1.4 Hz, J=7.7 Hz, 1H), 7.52-7.48 (m, 1H), 5.74-5.69(m, 1H), 5.47-5.39 (m, 1H), 5.26 (d, J=45.4 Hz, 2H), 3.52-3.44 (m, 2H),2.42-2.20 (m, 2H), 2.11-1.99 (m, 1H), 1.94-1.86 (m, 1H), 1.85 (d, J=3.3Hz, 3H), 1.83 (d, J=3.4 Hz, 3H), 1.34 (s, 9H). ¹³C NMR (100 MHz, CD₃ OD)δ 171.5, 165.3, 163.0, 161.8, 151.2, 136.3, 132.3, 131.7, 130.6, 130.2,129.8, 129.4, 127.4, 126.0, 125.4, 114.9, 114.7, 81.4, 78.5, 76.1, 51.0,45.1, 41.4, 28.7, 26.3, 23.7, 19.9, 19.8. HRMS m/z calculated forC₂₈H₃₆FN₅O₃(M+H⁺) 510.2875; found 510.2878.

(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-omithine)(9f)

¹H NMR (CD₃ OD; 400 MHz): δ 8.12-8.07 (m, 1H), 7.85-7.81 (m, 2H),7.63-7.57 (m, 3H), 7.55 (dd, J=1.3 Hz, J=7.8 Hz, 1H), 7.52-7.48 (m, 1H),5.73-5.68 (m, 1H), 5.43-5.46 (m, 1H), 4.37 (s, 2H), 3.47-3.42 (m, 2H),2.41-2.21 (m, 2H), 2.10-1.98 (m, 1H), 1.93-1.85 (m, 1H), 1.84 (d, J=3.1Hz, 3H), 1.82 (d, J=3.2 Hz, 3H), 1.36 (9H). ¹³C NMR (100 MHz, CD₃ OD) δ170.9, 168.8, 165.0, 163.1, 151.0, 136.4, 131.4, 130.5, 130.3, 129.7,129.3, 127.1, 125.6, 125.2, 115.1, 114.4, 81.5, 77.0, 76.1, 50.9, 45.5,41.8, 38.7, 28.9, 26.7, 23.4, 19.9, 19.8. HRMS m/z calculated forC₂₈H₃₆ClN₅O₃(M+H⁺) 526.2579; found 526.2577.

6. General Procedure for Synthesis of Benzimidazole haloacetamidines10a-h and 11a-h

To a stirred solution of 4a-d (1.0 eq) in THF was added5-phenylisobenzofuran-1,3-dione (1.0 eq) and allowed to stir at rt underN₂ for 18 h. Solvents were evaporated and the crude product was purifiedby reverse phase HPLC using MeCN:H₂O (0.5% TFA) as the eluent to givethe product in 78-86% yield. This product was then treated with TFA toremove the Boc group giving the 4-Ph-2-CO₂H-Bz-Orn-benzimidazole and3-Ph-2-CO₂H-Bz-Orn intermediates as a ˜50:50 mixture. The solvent wasthen evaporated to dryness and the crude material was dried in vacuo. Toa stirred solution of the corresponding 4-Ph-2-CO₂H-Bz-Orn-benzimidazoleand 3-Ph-2-CO₂H-Bz-Orn mixture in dry MeOH was added TEA (4.0 eq)followed by ethyl haloacetimidate HCl (2.0 eq). The reaction was stirredunder N₂ at rt for 3 h. Solvents were then evaporated under reducedpressure and the crude product was purified by reverse phase HPLC usingMeCN:H₂O (0.5% TFA) as an eluent to give compounds 10a-h and 11a-h in55-69% yield.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(10a) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-omithine)(11a)

Compounds 10a and 11a were isolated as a ˜40:60 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.27 (d, J=1.6 Hz, 0.4H), 8.14 (d, J=8.2 Hz,0.6H), 7.93 (dd, J=1.4 Hz, J=7.8 Hz, 0.4H), 7.86 (dd, J=1.5 Hz, J=7.9Hz, 0.6H), 7.81-7.63 (m, 5H), 7.54-7.48 (m, 4H), 7.43 (t, J=7.4 Hz, 1H),5.56-5.52 (m, 1H), 5.29 (d, J=45.3 Hz, 0.4H), 5.26 (d, J=45.4 Hz, 0.6H),3.52-3.45 (m, 2H), 2.38-2.21 (m, 2H), 2.07-1.87 (m, 2H). ¹³C NMR (125MHz, CD₃ OD) δ 171.8, 168.1, 167.9, 162.1, 154.7, 154.4, 154.2, 153.5,145.3, 142.8, 138.9, 138.8, 138.4, 133.6, 131.0, 130.2, 129.8, 128.8,128.5, 128.4, 128.1, 127.8, 126.9, 126.6, 126.0, 125.6, 124.8, 114.0,99.9, 78.3, 76.9, 67.3, 41.5, 28.9, 25.1, 23.5. HRMS m/z calculated forC₂₇H₂₆FN₅O₃(M+H⁺) 488.2092; found 488.2091.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-ornithine)(10b) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-omithine)(11b)

Compounds 10b and 11b were isolated as a ˜40:60 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.26 (d, J=1.9 Hz, 0.4H), 8.13 (d, J=8.2 Hz,0.6H), 7.93 (dd, J=2.1 Hz, J=8.1 Hz, 0.4H), 7.85 (dd, J=1.8 Hz, J=8.2Hz, 0.6H), 7.81-7.63 (m, 5H), 7.55-7.48 (m, 4H), 7.44 (J=7.06 Hz, 1H),5.56-5.52 (m, 1H), 4.39 (s, 0.8H), 4.37 (s, 1.2H), 3.49-3.43 (m, 2H),2.36-2.23 (m, 2H), 2.07-1.87 (m, 2H). ¹³C NMR (125 MHz, CD₃ OD) δ 171.9,171.8, 168.2, 167.8, 167.4, 163.2, 161.6, 161.1, 153.2, 145.1, 138.9,138.5, 138.0, 135.9, 132.3, 131.0, 130.3, 129.8, 128.8, 128.3, 127.8,127.3, 126.8, 126.6, 125.8, 125.3, 113.7, 41.9, 38.5, 28.9, 23.4, 20.2.HRMS m/z calculated for C₂₇H₂₆ClN₅O₃(M+H⁺) 504.1797; found 504.1798.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(10c) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(11c)

Compounds 10c and 11c were isolated as a ˜20:80 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.21 (d, J=2.1 Hz, 0.2H), 8.07 (d, J=8.1 Hz,0.8H), 7.92-7.78 (m, 3H), 7.72-7.57 (m, 5H), 7.50-7.38 (m, 2H),5.71-5.67 (m, 1H), 5.28 (d, J=45.3 Hz, 0.2H), 5.26 (d, J=45.4 Hz, 0.8H),4.24 (s, 3H), 3.55-3.43 (m, 2H), 2.43-2.33 (m, 1H), 2.31-2.21 (m, 1H),2.13-2.01 (m, 1H), 1.96-1.84 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.6,166.8, 163.1, 151.7, 145.1, 138.6, 137.6, 132.5, 130.8, 128.8, 128.3,127.8, 127.4, 126.8, 126.6, 126.3, 125.9, 125.7, 114.9, 114.3, 112.2,78.3, 76.6, 45.5, 41.3, 30.8, 28.3, 25.3. HRMS m/z calculated forC₂₈H₂₈FN₅O₃(M+H⁺) 502.2249; found 502.2255.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(10d) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(11d)

Compounds 10d and 11d were isolated as a ˜20:80 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (d, J=2.0 Hz, 0.2H), 8.08 (d, J=8.4 Hz,0.8H), 7.90-7.78 (m, 3H), 7.72-7.62 (m, 2H), 7.60-7.55 (m, 3H),7.51-7.38 (m, 2H), 5.70-5.66 (m, 1H), 4.39 (s, 1.6H), 4.38 (s, 0.4H),4.22 (s, 2.4H), 4.21 (s, 0.6H), 3.52-3.43 (m, 2H), 2.44-2.23 (m, 1H),2.11-1.85 (m, 1H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.9, 171.7, 168.1,167.8, 163.3, 161.7, 161.3, 153.3, 145.2, 142.8, 138.9, 138.7, 138.3,136.0, 132.2, 131.1, 130.2, 129.8, 128.7, 128.4, 128.3, 128.2, 128.0,127.9, 127.4, 126.8, 126.7, 125.9, 125.5, 113.8, 41.9, 38.7, 28.9, 23.5.HRMS m/z calculated for C₂₈H₂₈ClN₅O₃(M+H⁺) 518.1953; found 518.1957.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(10e) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(11e)

Compounds 10e and 11e were isolated as a ˜40:60 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (d, 0.4H), 8.07 (d, J=9.6 Hz, 0.6H),7.94-7.80 (m, 3H) 7.73-7.57 (m, 5H), 7.48 (t, J=8.0 Hz, 2H), 7.43-7.37(m, 1H), 5.69-5.65 (m, 1H), 5.27 (d, J=45.2 Hz, 0.8H), 5.26 (d, J=45.4Hz, 1.2H), 4.85-4.77 (m, 1H), 4.74-4.65 (m, 1H), 3.53-3.41 (m, 2H),2.42-2.30 (m, 1H), 2.29-2.19 (m, 1H), 2.13-2.01 (m, 1H), 1.96-1.84 (m,1H), 1.64 (t, J=8.0 Hz, 3H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.7, 171.5,167.3, 167.0, 163.0, 162.8, 161.4, 161.2, 151.6, 145.0, 142.9, 138.9,138.8, 137.8, 135.6, 131.6, 131.5, 130.9, 130.0, 129.8, 128.7, 128.3,128.3, 128.1, 128.0, 127.9, 127.6, 126.8, 126.6, 126.3, 126.0, 125.7,118.0, 115.1, 114.6, 112.4, 78.4, 76.7, 45.6, 41.4, 40.6, 29.0, 23.7,13.6. HRMS m/z calculated for C₂₉H₃₀FN₅O₃(M+H⁺) 516.2405; found516.2405.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(10f) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(11f)

Compounds 10f and 11f were isolated as a ˜40:60 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (d, J=2.3 Hz, 0.4H), 8.07 (dd, J=1.9Hz, J=9.6 Hz, 0.6H), 7.94-7.80 (m, 3H), 7.73-7.57 (m, 5H), 7.48 (t,J=7.4 Hz, 2H), 7.43-7.38 (m, 1H), 5.70-5.65 (m, 1H), 4.86-4.76 (m, 1H),4.73-4.65 (m, 1H), 4.39 (s, 0.8H), 4.38 (s, 1.2H),4.37 (s, 0.4H), 4.36(s, 0.6H), 3.50-3.40 (m, 2H), 2.41-2.31 (m, 1H), 2.29-2.20 (m, 1H),2.13-2.02 (m, 1H), 1.96-1.85 (m, 1H), 1.64 (t, J=7.5 Hz, 3H). ¹³C NMR(100 MHz, CD₃ OD) δ 171.7, 171.5, 167.4, 167.1, 163.3, 161.5, 161.2,151.7, 145.0, 142.9, 138.9, 138.8, 137.9, 135.7, 131.7, 130.9, 130.1,129.9, 128.8, 128.4, 128.3, 128.0, 128.0, 127.9, 127.6, 126.8, 126.6,126.3, 126.0, 125.8, 118.0, 115.1, 114.6, 112.4, 45.6, 45.5, 41.9, 40.6,38.7, 29.0, 28.9, 23.6, 13.6. HRMS m/z calculated for C₂₉H₃₀ClN₅O₃(M+H⁺)532.2110; found 532.2112.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(10g) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(11g)

Compounds 10g and 11g were isolated as a ˜40:60 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (d, J=2.2 Hz, 0.4H), 8.11-8.04 (m,1.6H), 7.90-7.79 (m, 2H), 7.72-7.63 (m, 3H), 7.60-7.54 (m, 2H),7.50-7.45 (m, 2H), 7.44-7.37 (m, 1H), 5.72-5.66 (m, 1H), 5.40-5.33 (m,1H), 5.27 (d, J=45.3 Hz, 0.4H), 5.21 (d, J=45.2 Hz, 0.6H), 3.53-3.43 (m,2H), 2.40-2.29 (m, 1H), 2.28-2.19 (m, 1H), 2.10-1.98 (m, 1H), 1.94-1.85(m, 1H), 1.85 (d, J=7.3 Hz, 6H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.6,171.3, 167.4, 167.2, 163.1, 161.4, 161.0, 161.4, 151.4, 145.0, 143.0,138.9, 138.8, 137.8, 135.6, 130.9, 130.4, 130.1, 130.0, 128.8, 128.4,128.3, 128.0, 128.0, 127.6, 126.8, 126.6, 125.8, 125.8, 125.4, 115.1,114.6, 78.4, 76.6, 51.1, 45.9, 45.8, 41.4, 29.2, 29.1, 23.7, 19.8, 19.8.HRMS m/z calculated for C₃₀H₃₂FN₅O₃(M+H⁺) 530.2562; found 530.2562.

(N1-[1,4′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(10h) and(N1-[1,3′-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(11h)

Compounds 10h and 11h were isolated as a ˜40:60 mixture, respectively.

¹H NMR (CD₃ OD; 400 MHz): δ 8.20 (d, J=2.2 Hz, 0.4H), 8.10-8.06 (m,1.6H), 7.90-7.80 (m, 2H), 7.72-7.64 (m, 3H), 7.60-7.55 (m, 2H),7.50-7.45 (m, 2H), 7.43-7.38 (m, 1H), 5.72-5.68 (m, 1H), 5.41-5.31 (m,1H), 4.38 (s, 0.8H), 4.37 (s, 1.2H), 3.49-3.41 (m, 2H), 2.41-2.31 (m,1H), 2.29-2.20 (m, 1H), 2.11-1.98 (m, 1H), 1.94-1.86 (m, 1H), 1.84-1.82(d, J=7.1 Hz, 6H). ¹³C NMR (100 MHz, CD₃ OD) δ 171.6, 167.5, 167.1,164.8, 163.3, 161.5, 161.0, 151.4, 146.7, 145.0, 142.9, 141.8, 138.8,137.9, 137.3, 135.6, 130.9, 130.5, 130.2, 130.0, 128.7, 128.4, 128.2,128.0, 127.9, 127.6, 126.8, 126.6, 125.7, 125.4, 115.1, 114.6, 110.0,51.0, 45.9, 45.8, 41.9, 38.7, 29.1, 23.6, 19.9, 19.8. HRMS m/zcalculated for C₃₀H₃₂FN₅O₃ (M+H⁺) 546.2266; found 546.2270.

Applicant's disclosure is described herein in preferred embodiments withreference to the Figures, in which like numbers represent the same orsimilar elements. Reference throughout this specification to “oneembodiment,” “an embodiment,” or similar language means that aparticular feature, structure, or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” and similar language throughout this specificationmay, but do not necessarily, all refer to the same embodiment.

The described features, structures, or characteristics of Applicant'sdisclosure may be combined in any suitable manner in one or moreembodiments. In the description herein, numerous specific details arerecited to provide a thorough understanding of embodiments of theinvention. One skilled in the relevant art will recognize, however, thatApplicant's composition and/or method may be practiced without one ormore of the specific details, or with other methods, components,materials, and so forth. In other instances, well-known structures,materials, or operations are not shown or described in detail to avoidobscuring aspects of the disclosure.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

1-37. (canceled)
 38. A molecular imaging probe having the structuralformula:A_(F)-L_(F)-W   (II) wherein A_(F) is a group comprising an opticallydetectable moiety; L_(F) is a linking group; and W is group comprising abenzimidazole moiety, or a derivative or analog thereof, capable ofinhibiting or inactivating a biological function of a protein argininedeiminase.
 39. The molecular imaging probe of claim 38, wherein A_(F) isa group comprising a fluorophore.
 40. The molecular imaging probe ofclaim 39, wherein the fluorophore is selected from the group consistingof xanthene dyes, cyanine dyes, coumarin dyes and bodipy dyes.
 41. Themolecular imaging probe of claim 40, wherein the fluorophore is axanthene dye selected from the group consisting of fluorescein, eosins,and rhodamines.
 42. The molecular imaging probe of claim 40 wherein thefluorophore is a cyanine dye.
 43. The molecular imaging probe of claim40, wherein the fluorophore is a coumarin dye.
 44. The molecular imagingprobe of claim 40, wherein the fluorophore is a bodipy dye.
 45. Themolecular imaging probe of claim 38, wherein the protein argininedeiminase is selected from the group consisting of: PAD1, PAD2, PAD3 andPAD4.
 46. The molecular imaging probe of claim 45, wherein the proteinarginine deiminase is PAD2.
 47. The molecular imaging probe of claim 45,wherein the protein arginine deiminase is PAD4.
 48. The molecularimaging probe of claim 38, wherein W is a monovalent radical derivedfrom a compound having the structural formula:

wherein, each of R_(a) and R_(b) is independently selected from thegroup consisting of H, D and F; L is a bivalent hydrocarbyl linker,optionally with one or more carbon atoms replaced by a heteroatomselected from the group consisting of O, S and N; X is a halogen atom; Yis N, O or S; provided that when Y is S or O, its bonding to theadjacent carbons are single bonds; Z is N—R₁, O or S; R₁ is selectedfrom the group consisting of: H, a C₁₋₆ alkyl, OH, a C₁₋₃ alkoxy, CF₃,COCH₃, and COCF₃ groups; each of R₂, R₃, R₄ and R₅ is independentlyselected from the group consisting of: H, hydroxyl, halogen atom, C₁₋₆alkyl, C₁₋₆ alkoxy, alkynyl, CF₂R_(c) and OCF₂R_(c) groups, where R_(c)is H, F or alkyl; and R₆ is a group comprising a cyclic alkyl or arylmoiety.
 49. A method for identifying a protein arginine deiminaseinhibitor or inactivator, comprising: performing a competitive assaywherein a test compound competes with a molecular imaging probeaccording to claim 38 to bind to a protein arginine deiminase; andmeasuring fluorescence to determine an amount of fluorescent proteinarginine deiminase present in the test assay.
 50. The method of claim49, further comprising: performing a control assay wherein the molecularimaging probe binds to the protein arginine deiminase; and measuringfluorescence to determine an amount of fluorescent protein argininedeiminase present in the control assay.
 51. The method of claim 50,wherein a change in fluorescence in the assay greater than apre-selected value when compared to the control assay is indicative thatthe test compound is an inhibitor to the protein arginine deiminase. 52.The method of claim 50, wherein the change in fluorescence in the assayis a decrease in fluorescence in the assay.